Combinations for the treatment of diseases involving cell proliferation, migration or apoptosis of myeloma cells, or angiogenesis

ABSTRACT

The present invention relates to a pharmaceutical combination for the treatment of diseases which involves cell proliferation, migration or apoptosis of myeloma cells, or angiogenesis. The invention also relates to a method for the treatment of said diseases, comprising co-administration of effective amounts of specific active compounds and/or co-treatment with radiation therapy, in a ratio which provides an additive and synergistic effect, and to the combined use of these specific compounds and/or radiotherapy for the manufacture of corresponding pharmaceutical combination preparations.

FIELD OF THE INVENTION

This invention relates to a method for the treatment of diseasesinvolving cell proliferation, migration or apoptosis of myeloma cells,or angiogenesis, which method comprises co-administration to a person inneed of such treatment and/or co-treatment of a person in need of suchtreatment with effective amounts of:

-   -   (i) a selected protein tyrosine kinase receptor antagonist; and    -   (ii) at least a further chemotherapeutic or naturally occurring,        semi-synthetic or synthetic therapeutic agent; and/or    -   (iii) radiotherapy or radio-immunotherapy.

This invention relates also to suitable pharmaceutical compositionscomprising effective amounts of:

-   -   (i) a selected protein tyrosine kinase receptor antagonist; and    -   (ii) at least a further chemotherapeutic or naturally occurring,        semi-synthetic or synthetic therapeutic agent;        and optionally adapted for a co-treatment with radiotherapy or        radio-immunotherapy, as a combined preparation for simultaneous,        separate or sequential use in the treatment of diseases        involving cell proliferation, migration or apoptosis of myeloma        cells, or angiogenesis, and especially for inhibiting tumour        growth, survival and metastasis.

This invention relates also to the combined use of effective amounts of:

-   -   (i) a selected protein tyrosine kinase receptor antagonist; and    -   (ii) at least a further chemotherapeutic or naturally occurring,        semi-synthetic or synthetic therapeutic agent;        for the manufacture of a pharmaceutical combined preparation for        simultaneous, separate or sequential use in the treatment of        diseases involving cell proliferation, migration or apoptosis of        myeloma cells, or angiogenesis, and especially for inhibiting        tumour growth, survival and metastasis, optionally in        combination with a co-treatment with radiotherapy or        radio-immunotherapy.

This invention relates also to the use of an effective amount of aselected protein tyrosine kinase receptor antagonist, for themanufacture of a pharmaceutical composition adapted for a simultaneous,separate or sequential co-treatment with radiotherapy orradio-immunotherapy of diseases involving cell proliferation, migrationor apoptosis of myeloma cells, or angiogenesis, and especially forinhibiting tumour growth, survival and metastasis.

BACKGROUND OF THE INVENTION

In the last decade, the biological activity of several types andsub-types of the protein tyrosin kinase receptor family have beencharacterised such as, for example, the epidermal growth factor receptorEGFR and its subtypes ErbB-2 and ErbB-4 (Brignola et al., Journal ofBiological Chemistry, Vol. 277, No. 2, pp. 1576-1585, 2002) or thevascular endothelial growth factor receptors VEGFR 1-3 together with itsligand VEGF and its four sub-types known to date (Jung et al., EuropeanJournal of Cancer, Vol. 38, pp. 1133-1140, 2002). Similar studiesreported in previous reports show that the overexpression of some ofthese receptors is implicated in multiple forms of cancer. For example,studies have provided evidence that the epidermal growth factor EGF actsas a growth factor in tumours, and that the vascular endothelial growthfactor VEGF is one of the most common mediators of tumor angiogenesis,which is essential for the growth and metastasis of solid tumours.Inhibitors of the receptors have thus been and are still evaluated forcancer therapy (see for example the article of Cerrington et al. InAdvances in Cancer Research, Academic Press 2000, pp. 1-38).

Recent studies have also suggested to combine several receptorantagonists together, or in further combination with a chemotherapeuticagent or radiation. For example, WO 02/070008 suggests the combinationof an antagonist specifically directed against the VEGF receptor with anantagonist specifically directed against the EGF receptor, optionallytogether with radiation or a chemotherapeutic agent, for the inhibitionof tumour growth. As example of suitable specific antagonists, WO02/070008 discloses monoclonal antibodies directed against the VEGFreceptor and monoclonal antibodies directed against the EGF receptor.

Thus, a large number of protein tyrosine kinase receptor antagonists arecurrently in clinical development for the treatment of cancer (see forexample the Expert Opinion Review of Laid & Cherrington in Expert Opin.Invest. Drugs, Vol. 12, No. 1, pp. 51-64, 2003). However, proof ofefficacy for these substances, used alone or with other cancertherapies, in the treatment of oncological diseases, has so far not beenachieved, either because of a lack of additional benefit over thestandard therapy or because of the discovery of unacceptableside-effects.

For example, it has been recently published that an angiogenesisinhibitor which has already been clinically tested, also in conjunctionwith chemotherapy, namely the inhibitor with code name SU5416, developedby Pharmacia for the treatment of cancer, was associated with disturbingside effect, namely thromboembolic events (Ken Garber and Ann Arbor,Nature Biotechnology, Vol. 20, pp. 1067-1068, November 2002).

For the treatment of diseases of oncological nature, a large number ofchemotherapeutic agents have already been suggested, which can be usedas mono-therapy (treatment with one agent) or as combination therapy(simultaneous, separate or sequential treatment with more than oneagent) and/or which may be combined with radiotherapy orradio-immunotherapy. In this respect, chemotherapeutic agent means anaturally occurring, semi-synthetic or synthetic chemical compoundwhich, alone or via further activation, for example with radiations inthe case of radio-immunotherapy, inhibits or kills growing cells, andwhich can be used or is approved for use in the treatment of diseases ofoncological nature, which are commonly also denominated as cancers. Inthe literature, these agents are generally classified according to theirmechanism of action. In this matter, reference can be made, for example,to the classification made in “Cancer Chemotherapeutic Agents”, AmericanChemical Society, 1995, W. O. Foye Ed.

Thus, within the meaning of the present invention, the following classesof chemotherapeutic agents are especially of interest, although notrepresenting a limitation:

-   -   Synthetic small molecule VEGF receptor antagonists    -   Small molecule growth factor (GF) receptor antagonists    -   Inhibitors of the EGF receptor and/or VEGF receptor and/or        integrin receptors or any other protein tyrosine kinase        receptors, which are not classified under the synthetic        small-molecules    -   Inhibitors directed to EGF receptor and/or VEGF receptor and/or        integrin receptors or any other protein tyrosine kinase        receptors, which are fusion proteins    -   Compounds which interact with nucleic acids and which are        classified as alkylating agents or platinum compounds    -   Compounds which interact with nucleic acids and which are        classified as anthracyclines, as DNA intercalators or as DNA        cross-linking agents    -   Anti-metabolites    -   Naturally occurring, semi-synthetic or synthetic bleomycin type        antibiotics (BLM-group antibiotics)    -   Inhibitors of DNA transcribing enzymes, especially topoisomerase        I or topoisomerase II inhibitors    -   Chromatin modifying agents    -   Mitosis inhibitors, anti-mitotic agents, or cell-cycle        inhibitors    -   Proteasome inhibitors    -   Enzymes    -   Hormones, hormone antagonists or hormone inhibitors, or        inhibitors of steroid biosynthesis    -   Steroids    -   Cytokines, hypoxia-selective cytotoxins, inhibitors of        cytokines, lymphokines, antibodies directed against cytokines or        oral and parenteral tolerance induction strategies    -   Supportive agents    -   Chemical radiation sensitizers and protectors    -   Photochemically activated drugs    -   Synthetic poly- or oligonucleotides    -   Other chemotherapeutic or naturally occurring, semi-synthetic or        synthetic therapeutic agents, such as cytotoxic antibiotics,        antibodies targeting surface molecules of cancer cells,        inhibitors of metalloproteinases, inhibitors of oncogenes,        inhibitors of gene transcription or of RNA translation or        protein expression, or complexes of rare earth elements

Further classes of compounds, so-far not classified as chemotherapeuticagents, which are naturally occurring, semi-synthetic or synthetictherapeutic agents, such as the non-steroidal anti-inflammatory drugs,especially the cyclooxygenase (COX) inhibitors and more specifically theCOX-2 inhibitors, are also of interest for combination therapies.

Even if the concept of combining several therapeutic agents or therapiesalready has been suggested, and although various combination therapiesare under investigation and in clinical trials, there is still a needfor new and efficient therapeutic agents for the treatment of diseasesin which cell proliferation, migration or apoptosis of myeloma cells, orangiogenesis, and there is still a need to develop further combinationswhich can show increased efficacy and reduced side-effects.

These diseases may as well be of oncological nature, which includes alltypes of malignant neoplasias or cancers, or of non-oncological nature,such as diabetic retinopathy, rheumatoid arthritis or psoriasis.

SUMMARY OF THE INVENTION

It has now been found that co-administration to a person in need of suchtreatment and/or co-treatment of a person in need of such treatment witheffective amounts of

-   -   (i) a selected protein tyrosine kinase receptor antagonist, and    -   (ii) at least a further chemotherapeutic or naturally occurring,        semi-synthetic or synthetic therapeutic agent, and/or    -   (iii) radiotherapy or radioimmunotherapy,        provides unexpected advantages in the treatment of diseases in        which cell proliferation, migration or apoptosis of myeloma        cells, or angiogenesis are involved, to a person in need of such        treatment, with high efficacy, in comparison to administration        of any of these substances alone and/or treatment with        radiotherapy or radioimmunotherapy.

It has been further found that this co-administration or co-treatment isespecially efficient if the selected protein tyrosine kinase receptorantagonist is an antagonist of at least one receptor selected fromVEGFR1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR, HER2, IGF1R, HGFR orc-Kit.

It has been further found that this co-administration or co-treatment isespecially efficient if the selected protein tyrosine kinase receptorantagonist is an antagonist of at least one receptor selected from VEGFR1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR, HER2, IGF1R, HGFR or c-Kit,and further an antagonist of a src tyrosine kinase family member, andespecially of src, lck, lyn and fyn, and/or further an antagonist of atleast one complex of a cyclin dependent kinase with its specific cyclinor with a viral cyclin, such as CDK1, CDK2, CDK3, CDK4, CDK5, CDK6,CDK7, CDK8 and CDK9 with their specific cyclins A, B1, B2, C, D1, D2,D3, E, F, G1, G2, H, I and K, and/or further an inhibitor of theparacrine IL-6 secretion.

Further it has been found that the diseases which can be treated by thecombination in accordance with the present invention are all kind ofdiseases in which cell proliferation, migration or apoptosis of myelomacells, or angiogenesis are involved, which can be of oncological naturesuch as all types of malignant neoplasias or cancers, or ofnon-oncological nature, such as diabetic retinopathy, rheumatoidarthritis, or psoriasis.

Further it has been found that the combination treatment in accordancewith the present invention is especially efficient for inhibiting tumourgrowth, survival and metastasis.

Further it has been found that the combination treatment in accordancewith the present invention is especially efficient with selected activesubstances, selected dosages and selected dosage forms.

Thus, the present invention provides a method for the treatment ofdiseases involving cell proliferation, migration or apoptosis of myelomacells, or angiogenesis, which method comprises simultaneous, separate orsequential co-administration of effective amounts of:

-   -   (i) an antagonist of at least one receptor selected from VEGFR 1        to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR, HER2, IGF1R, HGFR or        c-Kit, which is further an antagonist of a src tyrosine kinase        family member, or a polymorph, metabolite or pharmaceutically        acceptable salt thereof; and    -   (ii) at least a further chemotherapeutic or naturally occurring,        semi-synthetic or synthetic therapeutic agent;        in the form of a combined preparation, optionally adapted for a        co-treatment with radiotherapy or radio-immunotherapy, to a        person in need of such treatment.

The present invention provides also a method for the treatment ofdiseases involving cell proliferation, migration or apoptosis of myelomacells, or angiogenesis, which method comprises a simultaneous, separateor sequential co-treatment with an effective amount of an antagonist ofat least one receptor selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2and 3, EGFR, HER2, IGF1R, HGFR or c-Kit, which is further an antagonistof a src tyrosine kinase family member, or with a polymorph, metaboliteor pharmaceutically acceptable salt thereof, and with radiotherapy orradio-immunotherapy.

The protein tyrosine kinase receptor antagonist used in the method inaccordance with the present invention is preferably an antagonist of atleast one receptor selected from VEGFR 1 to 3, PDGFR α and β, FGFR1, 2and 3, EGFR, HER2, IGF1R, HGFR, c-Kit, and further an antagonist of asrc-tyrosine kinase family member, and especially of src, lck, lyn orfyn.

In a further preferred embodiment, the protein tyrosine kinase receptorantagonist may further be an antagonist of at least one complex of acyclin dependent kinase with its specific cyclin or with a viral cyclin,such as CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8 and CDK9 withtheir specific cyclins A, B1, B2, C, D1, D2, D3, E, F, G1, G2, H, I andK, and/or further an inhibitor of the paracrine IL-6 secretion.

In one preferred embodiment, the protein tyrosine kinase receptorantagonist is selected from specific compounds.

The further chemotherapeutic or naturally occurring, semi-synthetic orsynthetic therapeutic agent used in the method in accordance with thepresent invention can be any available chemotherapeutic or naturallyoccurring, semi-synthetic or synthetic therapeutic agent, and moreparticularly the chemotherapeutic agents which are commonly used for thetreatment of cancer. Preferred chemotherapeutic agents are selected fromthe following groups: synthetic small molecule VEGF receptorantagonists, small molecule growth factor (GF) receptor antagonists,inhibitors of the EGF receptor and/or VEGF receptor and/or integrinreceptors or any other protein tyrosine kinase receptors which are notclassified under the synthetic small-molecules, inhibitors directed toEGF receptor and/or VEGF receptor and/or integrin receptors or any otherprotein tyrosine kinase receptors, which are fusion proteins, compoundswhich interact with nucleic acids and which are classified as alkylatingagents or platinum compounds, compounds which interact with nucleicacids and which are classified as anthracyclines, as DNA intercalators(including DNA minor-groove binding compounds) or as DNA cross-linkingagents, anti-metabolites, naturally occurring, semi-synthetic orsynthetic bleomycin type antibiotics (BLM-group antibiotics), inhibitorsof DNA transcribing enzymes, and especially the topoisomerase I ortopoisomerase II inhibitors, chromatin modifying agents, mitosisinhibitors, anti-mitotic agents, cell-cycle inhibitors, proteasomeinhibitors, enzymes, hormones, hormone antagonists or hormoneinhibitors, or inhibitors of steroid biosynthesis, steroids, cytokines,hypoxia-selective cytotoxins, inhibitors of cytokines, lymphokines,antibodies directed against cytokines or oral and parenteral toleranceinduction strategies, supportive agents, chemical radiation sensitizersand protectors, photochemically activated drugs, synthetic poly- oroligonucleotides, optionally modified or conjugated, non-steroidalanti-inflammatory drugs, cytotoxic antibiotics, antibodies targetingsurface molecules of cancer cells, inhibitors of metalloproteinases,metals, inhibitors of oncogenes, inhibitors of gene transcription or ofRNA translation or protein expression, complexes of rare earth elements,or photo-chemotherapeutic agents.

In one preferred embodiment, amongst the chemotherapeutic or naturallyoccurring, semi-synthetic or synthetic therapeutic agents, specificcompounds are preferred.

In one embodiment, the disease treated in the method in accordance withthe present invention is preferably an oncological disease. In apreferred embodiment, the disease is selected from solid tumours, suchas urogenital cancers (such as prostate cancer, renal cell cancers,bladder cancers), gynecological cancers (such as ovarian cancers,cervical cancers, endometrial cancers), lung cancer, gastrointestinalcancers (such as colorectal cancers, pancreatic cancer, gastric cancer,oesophageal cancers, hepatocellular cancers, cholangiocellular cancers),head and neck cancer, malignant mesothelioma, breast cancer, malignantmelanoma or bone and soft tissue sarcomas, and haematologic neoplasias,such as multiple myeloma, acute myelogenous leukemia, chronicmyelogenous leukemia, myelodysplastic syndrome and acute lymphoblasticleukemia. In a preferred embodiment, the disease is hormone sensitive orhormone refractory prostate cancer, ovarian carcinoma, or small celllung cancer.

In another embodiment, the disease treated in the method in accordancewith the present invention is preferably a non-oncological diseaseselected from diabetic retinopathy, rheumatoid arthritis or psoriasis.

Thus, the beneficial efficacy of the methods in accordance with theinvention are mainly based on the additive and synergistic effects ofthe combined treatment, or to an improved tolerability of the treatmentby the patient due, for example, to the administration of lower doses ofthe therapeutic agents involved.

The unexpected advantages mentioned above may also be due to a moreefficient apoptosis induction by the chemotherapeutic agent, once theconstitutively active survival signal of the protein tyrosin kinasereceptor, mediated by the tumour, is inhibited by the selected proteintyrosine kinase receptor antagonist.

In the case of the use of an antagonist of protein tyrosine kinasereceptors or an inhibitor of other mediators involved in angiogenesis,such as for example the vascular endothelial growth factors (VEGF), thevascular permeability factors, the basic fibroblast growth factor(bFGF), interleukin-6 (IL-6) or interleukin-8 (IL-8), the epidermalgrowth factor (EGF) or the platelet-derived growth factor (PDGF), one ofthe advantages of the method and composition in accordance with thepresent invention lies in a targeting of the treatment totumour-associated vasculature rather than, or together with, the tumouritself, in order to cut the energy supply of cancerous cells.

A further advantage is that an induction or reinstatement of thesensitivity towards the chemotherapeutic agent is expected in patientstreated with the combination of chemotherapeutic agents for which thesensitivity gets lost in the course of the treatment and of a VEGFRantagonist. This is especially the case of patients suffering fromrefractory multiple myeloma and treated with steroids aschemotherapeutic agent. A combination treatment with steroids and aVEGFR antagonist is expected to restore the steroid sensitivity ofpatients suffering from refractory multiple myeloma.

According to the present invention, a synergistic combined preparationis meant to comprise an amount of the selected protein tyrosine kinasereceptor antagonist, or of a polymorph, metabolite or pharmaceuticallyacceptable salt of this active compound, and an amount of the furtherchemotherapeutic or naturally occurring, semi-synthetic or synthetictherapeutic agent, and/or radiotherapy or radio-immunotherapy, whereinthe amount of the individual therapeutic agents alone is insufficient toachieve the therapeutic effect achieved by the administration of thecombination of said therapeutic agents, and wherein the combined effectsof the amounts of the therapeutic agents is greater than the sum of thetherapeutic effects achievable with the amounts of the individualtherapeutic agents.

Viewed from a different aspect, the present invention also relates to apharmaceutical combination for the treatment of diseases in which cellproliferation, migration or apoptosis of myeloma cells, or angiogenesisare involved, comprising a selected specific protein tyrosine kinasereceptor antagonist and a further chemotherapeutic or naturallyoccurring, semi-synthetic or synthetic therapeutic agent, and/orradiotherapy or radio-immunotherapy, as a combined preparation forsimultaneous, separate or sequential use in treatment of said diseases,optionally together with one or more pharmaceutically acceptablediluents and/or carriers.

Viewed from a different aspect, the present invention also relates to apharmaceutical combination preparation kit for the treatment of diseasesinvolving cell proliferation, migration or apoptosis of myeloma cells,or angiogenesis, comprising a therapeutically effective amount of aselected protein tyrosine kinase receptor antagonist, or of a polymorph,metabolite or pharmaceutically acceptable salt thereof, and atherapeutically effective amount of a further chemotherapeutic ornaturally occurring, semi-synthetic or synthetic therapeutic agent,characterised in that the protein tyrosine kinase receptor antagonist iscomprised within a first compartment and the further chemotherapeutic ornaturally occurring, semi-synthetic or synthetic therapeutic agent iscomprised within a second compartment, such that the administration to apatient in need thereof can be simultaneous, separate or sequential,said combination preparation kit being optionally further adapted for aco-treatment with radiotherapy or radio-immunotherapy.

In one embodiment in accordance with the present invention, in eachcompartment of the pharmaceutical combination preparation kit, eachactive substance is formulated for an oral administration.

Viewed from a further aspect, the present invention thus also providesthe use of a selected protein tyrosine kinase receptor antagonist incombination with a further chemotherapeutic or naturally occurring,semi-synthetic or synthetic therapeutic agent, and/or adapted for aco-treatment with radiotherapy or radio-immunotherapy, for themanufacture of a pharmaceutical combination preparation for thetreatment of the diseases or indications mentioned hereinbefore.

Within the meaning of the present invention, effective amounts oftherapeutic agents and/or of a therapeutic treatment by radiotherapy orradio-immunotherapy means amounts of the agents and/or of the treatmentby radiotherapy or radio-immunotherapy which are effective to achieve atherapeutic effect when used in combination.

DETAILED DESCRIPTION OF THE INVENTION

The Diseases

As already mentioned hereinbefore, the diseases which can be treated bythe combination in accordance with the present invention are all kind ofdiseases in which cell proliferation, migration or apoptosis of myelomacells, or angiogenesis are involved, which can be of oncological naturesuch as all types of malignant neoplasias or cancers, or ofnon-oncological nature, such as diabetic retinopathy, rheumatoidarthritis, or psoriasis. Among cancers, selected specific targetindications are solid tumours, such as urogenital cancers (such asprostate cancer, renal cell cancers, bladder cancers), gynecologicalcancers (such as ovarian cancers, cervical cancers, endometrialcancers), lung cancer, gastrointestinal cancers (such as colorectalcancers, pancreatic cancer, gastric cancer, oesophageal cancers,hepatocellular cancers, cholangiocellular cancers), head and neckcancer, malignant mesothelioma, breast cancer, malignant melanoma orbone and soft tissue sarcomas, and haematologic neoplasias, such asmultiple myeloma, acute myelogenous leukemia, chronic myelogenousleukemia, myelodysplastic syndrome and acute lymphoblastic leukemia.

The combination treatment in accordance with the present invention isespecially efficient for inhibiting tumour growth, survival andmetastasis.

Of special interest for the combination treatment is the treatment ofhormone sensitive or hormone refractory prostate cancer, ovariancarcinoma, non small cell lung cancer, small cell lung cancer, ormultiple myeloma.

The Selected Protein Tyrosine Kinase Receptor Antagonist

As already mentioned hereinbefore, the selected protein tyrosine kinasereceptor antagonists that can be used in the context of the presentinvention include all substances that inhibit the stimulation oractivation of a protein tyrosine kinase receptor by a protein tyrosinekinase receptor ligand. In the case of a protein tyrosine kinasereceptor belonging to the family of the growth factor receptors, suchinhibition of stimulation or activation inhibits the growth of cellsthat express the receptor.

Some examples of growth factor receptors involved in tumorigenesis arethe receptors for epidermal growth factor (EGFR), vascular endothelialgrowth factors (VEGFRs), platelet-derived growth factor (PDGFR),insulin-like growth factor (IGFR), nerve growth factor (NGFR), andfibroblast growth factor (FGFR).

By inhibition of stimulation or activation of protein tyrosine kinasereceptor is meant any decrease in the activation of the receptor, whichneed not completely prevent or stop activation of the receptor.

Moreover, inhibition of the receptor stimulation or activation, asdefined by the present invention, means inhibition resulting frominteraction of the antagonist and the receptor or its ligand. Byinteraction is meant sufficient physical or chemical interaction betweenthe antagonist and the receptor, such that protein tyrosin kinaseactivity is inhibited. One of skill in the art would appreciate thatexamples of such chemical interactions, which include association orbonding, are known in the art and include covalent bonding, ionicbonding, hydrogen bonding, etc. . . . , between the antagonist and thereceptor or its ligand.

Increased protein tyrosine kinase receptor stimulation or activation canresult from higher levels of ligand, receptor gene amplification,increased transcription of the receptor or mutations that causeunregulated receptor signalling. Amplification of the gene encoding thereceptor results in an increased number of ligands binding to thereceptor, which can further stimulate cell proliferation. The proteintyrosine kinase receptor may also be over-expressed in the absence ofgene amplification, presumably through mutations that increase itstranscription, mRNA translation, or stability of the protein. Proteintyrosine kinase receptor mutants of the EGFR type have already beenidentified in gliomas, non-small cell lung carcinomas, ovariancarcinomas and prostate carcinomas, that have a constitutively activeprotein tyrosin kinase, suggesting a role for high-level EGFR activityrather than EGFR over-expression in these cancers (see for examplePedersen et al., Ann. Oncol., Vol. 12(6), pp. 745-60, 2001).

In one embodiment in accordance with the present invention, the selectedprotein tyrosine kinase receptor antagonist inhibits the binding of theprotein tyrosine kinase receptor to its ligand.

Binding of a ligand to an external, extracellular domain of the receptorstimulates receptor dimerization, autophosphorylation of the receptor,activation of the receptor's internal, cytoplasmic protein tyrosinkinase domain, and initiation of multiple signal transduction pathwaysinvolved in regulation of DNA synthesis, cell division, vasculogenesisor angiogenesis. The inhibition produced by the presence of theantagonist will consequently reduce this stimulation.

In another embodiment in accordance with the present invention, theselected protein tyrosine kinase receptor antagonist binds directly tothe receptor. The antagonist can bind externally to the extra-cellularportion of the receptor, which may or may not inhibit binding of theligand, or internally to the protein tyrosine kinase domain. Examples ofsuch antagonists include, without limitation, biological molecules, suchas antibodies (and functional equivalents thereof) specific for thereceptor, and synthetic kinase inhibitors that act directly on thecytoplasmic domain of the receptor, such as the so-called “smallmolecule tyrosine kinase inhibitors”. A non-exhaustive list of smallmolecule tyrosine kinase inhibitors can be found in the review articleof Laid & Cherrington, Expert Opinion Invest. Drugs, Vol. 12, No. 1,2003, the content of which is incorporated herein by reference.

Additional protein tyrosine kinase receptor antagonists can easily bedetermined using well-known methods. The selected receptor antagoniststo be used in the present invention inhibit the protein tyrosin kinaseactivity of the receptor, which generally involves phosphorylationevents. Accordingly, phosphorylation assays may for example be useful indetermining antagonists useful in the context of the present invention.In addition, methods specific for detection of the receptor expressioncan be utilized. These include immunohistochemistry for detection ofprotein expression, fluorescence in situ hybridization for detection ofgene amplification, competitive radioligand binding assays, solid matrixblotting techniques, such as Northern and Southern blots, reversetranscriptase polymerase chain reaction and ELISA.

In accordance with the present invention, the selected protein tyrosinekinase receptor antagonist is preferably an antagonist of at least onereceptor selected from VEGFR 1 to 3, PDGFR α and β, FGFR1,2 and 3, EGFR,HER2, IGF1R, HGFR, c-Kit, and further an antagonist of one of thesrc-tyrosine kinase family members, and especially src, lck, lyn or fyn,or a polymorph, metabolite or pharmaceutically acceptable salt thereof.The selected protein tyrosine kinase receptor antagonist may further bean antagonist of at least one complex of a cyclin dependent kinase withits specific cyclin or with a viral cyclin, such as CDK1, CDK2, CDK3,CDK4, CDK5, CDK6, CDK7, CDK8 and CDK9 with their specific cyclins A, B1,B2, C, D1, D2, D3, E, F, G1, G2, H, I and K, and/or further an inhibitorof the paracrine IL-6 secretion.

In a further embodiment in accordance with the present invention, thecombination of the active substances is intended for the treatment ofoncological diseases involving angiogenesis.

Tumour angiogenesis plays an important role in the progression of humanmalignancies. Inhibition of this process is thought to be an excellentpoint of therapeutic intervention in the treatment of cancer. Signaltransduction through the vascular endothelial growth factor receptor 2(VEGFR-2) has been shown to play a pivotal role in the proliferation,survival and migration of endothelial cells in tumour angiogenesis.

In this matter, potent and orally available low molecular weightantagonists of VEGFR-2 have been developed as new compounds which areuseful for the treatment of diseases involving cell proliferation,migration or apoptosis of myeloma cells, or angiogenesis, and especiallyas new cancer therapeutic agents. These antagonists are thus inhibitorsof the activity of the receptor. Some of these antagonists are alsoantagonists of further growth factor receptors, such as VEGFR-3, PDGFR αand β, FGFR1, 2 and 3, EGFR, HER2, IGF1R, HGFR, c-Kit, and some alsoantagonists of the src-tyrosine kinase family members src, lck, lyn andfyn.

These compounds are disclosed in WO 02/36564, WO 99/52869, WO 00/18734,WO 00/73297, WO 01/27080, WO 01/27081 and WO 01/32651 The citeddocuments are herewith incorporated by reference with respect to anyaspects disclosed relating to these specific compounds.

The following compounds are particularly representative and are allcombined inhibitors of VEGFR-2 and lck which may be used as the selectedprotein tyrosine kinase receptor antagonist within the meaning of thepresent invention.

-   (A)    (Z)-3-(1-(4-(N-(2-dimethylamino-ethyl)-N-methylsulfonyl-amino)-phenylamino)-1-phenyl-methylene)-2-indolinone;-   (B)    (Z)-3-(1-(4-(N-(3-dimethylaminopropyl)-N-propionyl-amino)-phenylamino)-1-phenyl-methylene)-2-indolinone;-   (C)    (Z)-3-(1-(4-(dimethylaminomethyl)-phenylamino)-1-phenyl-methylene)-5-(butylcarbamoyl)-2-indolinone;-   (D)    (Z)-3-(1-(4-(dimethylaminomethyl)-phenylamino)-1-phenyl-methylen)-5-(cyclohexylmethyl-carbamoyl)-2-indolinone;-   (E)    (Z)-3-(1-(4-(N-methylsulfonyl-N-(2-dimethylamino-ethyl)-amino)-phenylamino)-1-phenyl-methylen)-5-(cyclohexylmethyl-carbamoyl)-2-indolinone;-   (F)    (Z)-3-(1-(4-(butylaminomethyl)-phenylamino)-1-phenyl-methylen)-5-(cyclohexylmethyl-carbamoyl)-2-indolinone;-   (G)    (Z)-3-(1-(4-(pyrrolidin-1-yl-methyl)-phenylamino)-1-phenyl-methylen)-5-(cyclohexylmethyl-carbamoyl)-2-indolinone;-   (H)    (Z)-3-(1-(4-(diethylaminomethyl)-phenylamino)-1-phenyl-methylen)-5-(cyclohexylmethyl-carbamoyl)-2-indolinone;-   (I)    (Z)-3-(1-(4-(diethylaminomethyl)-phenylamino)-1-phenyl-methylen)-5-(N-(3-chlorobenzyl)-carbamoyl)-2-indolinone;-   (J)    (Z)-3-(1-(4-(diethanolaminomethyl)-phenylamino)-1-phenyl-methylen)-5-(butylcarbamoyl)-2-indolinone;-   (K)    (Z)-3-(1-(4-(dimethylaminomethyl)-phenylamino)-1-phenyl-methylen)-5-(N-(3-chlorobenzyl)-carbamoyl)-2-indolinone;-   (L)    (Z)-3-(1-(4-(N-acetyl-N-(2-dimethylamino-ethyl)-amino)-phenylamino)-1-phenyl-methylen)-5-(N-(3-chlorobenzyl)-carbamoyl)-2-indolinone;-   (M)    (Z)-3-(1-(4-(butylaminomethyl)-phenylamino)-1-phenyl-methylen)-5-(N-(3-chlorobenzyl)-carbamoyl)-2-indolinone;-   (N)    (Z)-3-(1-(4-(dimethylaminomethyl)-phenylamino)-1-phenyl-methylene)-6-methoxycarbonyl-2-indolinone;-   (O)    (Z)-3-(1-(4-(N-(3-dimethylamino-propyl)-N-acetyl-amino)-phenylamino)-1-phenyl-methylene)-6-methoxycarbonyl-2-indolinone;-   (P)    (Z)-3-(1-(4-(ethylaminomethyl)-phenylamino)-1-phenyl-methylene)-6-methoxycarbonyl-2-indolinone;-   (Q)    (Z)-3-(1-(4-(1-methyl-imidazol-2-yl)-phenylamino)-1-phenyl-methylene)-6-methoxycarbonyl-2-indolinone;-   (R)    (Z)-3-(1-(4-(N-(dimethylaminomethylcarbonyl)-N-methyl-amino)-phenylamino)-1-phenyl-methylene)-6-methoxycarbonyl-2-indolinone;-   (S)    (Z)-3-(1-(4-(methylaminomethyl)-anilino)-1-phenyl-methylene)-6-methoxycarbonyl-2-indolinone;-   (T)    (Z)-3-(1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-phenylamino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone;    and-   (U)    4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)-quinazoline,    as well as their polymorphs, metabolites or pharmaceutically    acceptable salts.

Compounds (A) to (B) are described in WO 00/18734, compounds (C) to (M)are described in WO 00/73297, compounds (N) to (T) are described in WO01/27081, compound (U) is described in WO 01/32651.

Especially representative is the potent and orally available lowmolecular weight antagonist of VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and3, EGFR, HER2, IGF1R, HGFR and c-Kit, which is further an antagonist ofthe src tyrosine kinase family members, and especially of src, lck, lynand fyn, further an antagonist of the complex of cyclin dependentkinases with their specific cyclins or with a viral cyclin, and furtheran inhibitor of the paracrine IL-6 secretion, disclosed, for example, inWO 01/27081, as exemplified compound number 473, as well as itspolymorphs, metabolites or pharmaceutically acceptable salts. Thiscompound, referred to as (T) in the above list, is3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone.

When compared to the other above exemplified compounds, this compound isfurther particularly preferred due to its high potency as inhibitor andits better toxicologic profile.

Particularly preferred is the monoethanesulfonate salt of this compound,namely the monoethanesulfonate salt of3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone,disclosed for example in unpublished German patent application DE 102 33500.1, unpublished PCT/03/07822 and unpublished U.S. patent applicationSer. No. 10/623,971.

In accordance with what is disclosed in DE 102 33 500.1, unpublishedPCT/03/07822 and unpublished U.S. patent application Ser. No.10/623,971, the monoethanesulfonate salt of3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinonehas the following chemical structure:

This compound may be selectively obtained by a suitable choice ofmanufacturing conditions, preferably in its crystalline hemihydrateform.

This compound is characterised by a melting point of T=305±5° C.(determined by DSC=Differential Scanning Calorimetry, using aMettler-Toledo DSC82 apparatus; heating rate: 10 K/min).

For the manufacture of the monoethanesulfonate salt of3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone,a procedure in accordance with the following may be used.

The starting material used to prepare the monoethanesulfonate salt of3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinonemay be the free base3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone,which may be obtained in accordance with a method known from the priorart and described, for example, in WO 01/27081.

Thus, in a first step and in accordance with what is described in WO01/27081,3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinoneis prepared as follows.

10.5 g (30.0 mmol)1-acetyl-3-(1-ethoxy-1-phenylmethylene)-6-methoxycarbonyl-2-indolinone(prepared as described in WO 01/27081) and 8.60 g (33.0 mmol)N-[(4-methyl-piperazin-1-yl)-methylcarbonyl]-N-methyl-p-phenylendiamine(prepared as described in WO 01/27081) are dissolved in 80 mldimethylformamide and mixed for 1 hour at 80° C. After cooling, 6.50 mlpiperidine is added and the whole is further mixed for 2 hours at roomtemperature. Water is added, the liquid over the resulting precipitateis sucked up, and the precipitate is washed again with a low quantity ofwater. The residue is suspended in 200 ml methanol, the liquid is suckedup, and the remaining residue washed with cold water and diethylether.The resulting product is vacuum dried at 110° C.

-   Recovered product: 12.4 g (77% of theoretical value)-   IR-spectroscopy: 1610, 1655, 1711 cm⁻¹-   T_(Smp.)=253° C.-   Molecular formula: C₃₁H₃₃N₅O₄-   Electrospray-mass spectrometry: m/z=540 [M+H]⁺-   Element analysis:

calculated C 68.99 H 6.16 N 12.98 found C 68.32 H 6.29 N 12.85

In a second step, and in accordance with what what is disclosed in DE102 33 500.1, the monoethanesulfonate salt of3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinonewill be obtained as follows.

605 g (1.12 mol)3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinoneare suspended in 9 litres methanol and heated to 50° C. 183.7 g (1.121mol) of a 70% aqueous solution of ethanesulfonate is added. Theresulting solution is cooled to 40° C. and mixed with 4.5 litrester-butylmethylether. Cristallisation occurs after a few minutes. Inorder to achieve a complete precipitation, the whole is mixed for 16hours at room temperature. After cooling to a temperature of 10° C., theliquid is sucked up, the precipitate is washed with 2 litrester-butylmethylether and vacuum dried at 40° C.

-   Recovered product: 638 g (87.6% of theoretical value)-   T_(Smp.)=305±5° C. (DSC 10K/min)-   Purity (measured by HPLC): 99.4%-   Water content: 1.0 bis 2.0% (KF)

The monoethanesulfonate salt of3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinonecan be very easily dissolved in physiologically acceptablesolubilization agents.

Additionally, the compound MES(T) is orally bioavailable in mice.

The monoethanesulfonate salt of3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinoneinhibits the human VEGFR-2 kinase (huVEGFR-2) with an IC₅₀ of 21 nM, themurine VEGFR-2 kinase (huVEGFR-2) with an IC₅₀ of 13 nM, and theproliferation of VEGF stimulated endothelial cells (HUVEC: IC₅₀=9 nM,HSMEC: IC₅₀=12 nM).

Furthermore, FGFR-1 and PDGFRα, two members of the split kinase domainfamily of receptors important in angiogenic signaling, are additionallyinhibited by this compound with IC₅₀'s of 69 nM and 59 nM respectively.

The compound MES(T) is thus highly selective when tested against a panelof numerous different kinases, as shown in the following Table I.

TABLE I Kinase IC₅₀ [nM] huVEGFR-2 21 muVEGFR-2 13 VEGFR-3 13 InsR >4000IGF1R >1000 EGFR >50000 HER2 >50000 FGFR1 69 FGFR3 137 PDGFRα 59CDK1 >10000 CDK2 >10000 CDK4 >10000 Lck 16 Lyn 195 Src 156

Noteworthy is also that this specific antagonist shows a long lastinginhibition of the receptor VEGFR-2. On the molecular and cellular levela short exposure of the compound MES(T) to cells (e.g. endothelialcells) is enough to inhibit the activation of the receptor kinase itselfand downstream signalling molecules (e.g. the MAP kinase, MAPK) as wellas cellular proliferation for at least 32 h.

The results of the following experiment evidences this long-lastinginhibition effect. In order to determine the duration of the inhibitioninduced by MES(T) on the receptor, washout experiments were performed.HUVEC and NIH 3T3 KDR cells were exposed to MES(T) for a limited periodof time, MES(T) was washed away and cell proliferation (HUVEC) orVEGFR-2 activation/phosphorylation was analysed after various periods oftime. As shown in FIG. 1, the autophosphorylation of VEGFR-2 is blockedfor at least 32 h after a 1 hour exposure with 50 nM MES(T). After 8 h,24 h, and 32 h without MES(T), the cells were again stimulated with VEGFand the receptor activation was analysed. Even after 32 h no receptoractivation could be observed. This strongly suggests that MES(T)exhibits sustained effects on the receptor kinase even when the MES(T)plasma concentration are very low.

The results of the following in vivo xenograft experiment evidences theeffect on tumour cells of compound MES(T). In order to determine thiseffect, nude mice bearing subcutaneous FaDu tumours (FaDu tumours areconstituted of human squamous carcinoma cells) were orally treated withthe compound MES(T). As shown in FIG. 2, when the mice were treatedtwice weekly with a dose of 100 mg/kg, a reduction of tumour growth witha T/C (Tumour/Control) value of 31% can be seen. By increasing the doseto 200 mg/kg orally twice weekly an even better anti-tumour effect isexpected.

This indicates that this antagonist is particularly suitable for asequential co-administration and/or co-treatment with anotherchemotherapeutic or naturally occurring, semi-synthetic or synthetictherapeutic agent, and/or radiotherapy or radio-immunotherapy. Thescheduled treatment regimen with this antagonist may be, for example, analternate treatment one day on/one day off, one day on/two days off, oneweek on/one week off, or even two weeks on/two weeks off.

The monoethanesulfonate salt of3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinoneis thus clearly a potent and orally available VEGFR-2 kinase inhibitorand anti-tumour agent.

With regard to all aspects of the invention, suitable selected proteintyrosine kinase receptor antagonists are also the active in vivometabolites of the selected protein tyrosine kinase receptorantagonists. For example, an active in vivo metabolite of the proteintyrosine kinase receptor antagonist3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinonemay be the unesterified compound3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-carbonyl-2-indolinone.

Within the meaning of the present invention, all the above-exemplifiedcompounds, and especially the compound (T) and its monoethanesulfonatesalt MES(T), may also be used as mono-therapy for the treatment of theabove-mentioned diseases, namely all kind of diseases in which cellproliferation, migration or apoptosis of myeloma cells, or angiogenesisare involved, which can be of oncological nature such as all types ofmalignant neoplasias or cancers, or of non-oncological nature, such asdiabetic retinopathy, rheumatoid arthritis, or psoriasis. Among cancers,selected specific target indications for a mono-therapeutic treatmentare solid tumours, such as urogenital cancers (such as prostate cancer,renal cell cancers, bladder cancers), gynecological cancers (such asovarian cancers, cervical cancers, endometrial cancers), lung cancer,gastrointestinal cancers (such as colorectal cancers, pancreatic cancer,gastric cancer, oesophageal cancers, hepatocellular cancers,cholangiocellular cancers), head and neck cancer, malignantmesothelioma, breast cancer, malignant melanoma or bone and soft tissuesarcomas, and haematologic neoplasias, such as multiple myeloma, acutemyelogenous leukemia, chronic myelogenous leukemia, myelodysplasticsyndrome and acute lymphoblastic leukemia. Of special interest is thetreatment of hormone sensitive or hormone refractory prostate cancer,ovarian carcinoma, non small cell lung cancer, small cell lung cancer,or multiple myeloma. The above-exemplified compounds are especiallyefficient for inhibiting tumour growth, survival and metastasis.

The Further Chemotherapeutic or Naturally Occurring, Semi-Synthetic orSynthetic Therapeutic Agent

This compound may preferably be selected from the following classes andexamples of compounds, although this list is not to be considered aslimitative.

Synthetic Small Molecule VEGF Receptor Antagonists

Synthetic small molecule VEGF receptor antagonists of particularinterest are the antagonists of the VEGF receptor of type 2, which areas well antagonists of the basic fibroblast growth factor (bFGF) and ofthe platelet derived growth factor (PDGF) receptors. Representativecompounds are, for example, indolinone derivatives, such as thosedescribed in WO 02/36564, WO 99/52869, WO 00/18734, WO 00/73297, WO01/27080, WO 01/27081 and WO 01/32651. Further representative smallmolecule VEGF receptor antagonists are the compounds described in WO01/60814, WO 99/48868, WO 98/35958, and especially the compoundsvatalanib (PTK-787/ZK222584), SU-5416, SU-6668, SU-11248, SU-14813,AZD-6474, AZD-2171, CP-547632, CEP-7055, AG-013736, IM-842 (a dipeptideof L-Glutamyl and L-Tryptophan) or GW-786034.

Small Molecule Growth Factor (GF) Receptor Antagonists

Small molecule growth factor (GF) receptor antagonists of particularinterest are the antagonists of the protein tyrosin kinase (PTK)receptors, especially the antagonists of the epidermal growth factor(EGF) receptor, the dual antagonists of the epidermal growth factor(EGF) and of the human epidermal growth factor of type 2 (HE type 2)receptors or the antagonists of the mitogen-activated protein kinase(MAPK). Representative compounds which are dual EGFR and HER-2antagonists are, for example, the quinazoline derivatives disclosed inWO 00/78735 and WO 02/50043, gefitinib, erlotinib, CI-1033 and GW-2016.Representative compounds which are only EGFR antagonists are, forexample, iressa (ZD-1839), tarceva (OSI-774), PKI-166, EKB-569, HKI-272and herceptin. Representative compounds which are antagonists of themitogen-activated protein kinase (MAPK) are BAY-43-9006 (a Raf proteinkinase family inhibitor) and BAY-57-9006 (a Kdr tyrosine kinaseinhibitor).

A preferred compound in this class is the quinazoline derivativedisclosed in WO 02/50043 as exemplified compound of Example 1(10),namely4-[(3-chloro-4-fluorophenyl)amino]-6-{[4-(N,N-dimethylamino)-1-oxo-2-buten-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)-quinazoline,or the tautomers, the stereoisomers and the salts thereof, particularlythe physiologically acceptable salts thereof with inorganic or organicacids or bases. Most preferred is the di-maleic acid salt of thiscompound, which can easily be obtained in accordance with the followingprocedure. 6.0 kg (12.35 mol) of4-[(3-chloro-4-fluorophenyl)amino]-6-{[4-(N,N-dimethylamino)-1-oxo-2-buten-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)-quinazolineare heated up to 70° C. in 84 liter of ethanol. A solution of 2.94 kg(25.31 mol) maleic acid in 36 liter ethanol is added. At the beginningof crystallisation, the reaction mixture is cooled to 20° C. and stirredfor 2 hours. The reaction mixture is cooled to 0° C. and stirred for 3hours. The precipitate is suction filtered. The filter cake is washedwith 19 liter of ethanol and vacuum-dried at 40° C.

A further preferred compound in this class is the4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-(homomorpholin-4-yl)-1-oxo-2-buten-1-yl]amino}-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline,or the salts thereof. The chemical structural formula of this compoundis

This compound may be obtained in three steps using the followingmanufacturing conditions.

Preparation of the Starting Compound I:4-[(3-chloro-4-fluoro-phenyl)amino]-6-[(diethoxy-phosphoryl)-acetylamino]-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline

60.07 g of diethoxyphosphorylacetic acid are placed in 750 ml ofN,N-dimethylformamide and at ambient temperature combined with 48.67 gof N,N′-carbonyldiimidazole. After the development of gas has ceased90.00 g of4-[(3-chloro-4-fluoro-phenyl)amino]-6-amino-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazolineare added and the reaction mixture is stirred for about 4-5 hours atambient temperature until the reaction is complete. The reaction mixtureis then heated gently in the water bath and 750 ml of water are addedtwice. The thick suspension is stirred overnight and the next morninganother 350 ml of water are added. The suspension is cooled in the icebath, stirred for one hour and suction filtered. The filter cake iswashed again with 240 ml of N,N-dimethylformamide/water (1:2) and 240 mlof diisopropylether and dried at 40° C. in the circulating air dryer.

-   Yield: 117.30 g of (88% of theory)-   R_(f) value: 0.37 (silica gel, methylene chloride/methanol=9:1)-   Mass spectrum (ESI⁺): m/z=553, 555 [M+H]⁺

Preparation of the Starting Compound II:Homomorpholin-4-yl-acetaldehyde-hydrochloride

Prepared by stirring (2.5 hours) 4-(2,2-dimethoxy-ethyl)-homomorpholinewith semi-concentrated hydrochloric acid at 80° C. The solution obtainedis further reacted directly as below-described.

Preparation of the Final Compound:4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-(homomorpholin-4-yl)-1-oxo-2-buten-1-yl]amino}-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline

A solution of 3.9 g of4-[(3-chloro-4-fluoro-phenyl)amino]-6-[2-(diethoxy-phosphoryl)-acetylamino]-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline(starting compound I) in 20 ml of tetrahydrofuran is added to a solutionof 300 mg of lithium chloride in 20 ml of water at ambient temperature.Then 2.35 g of potassium hydroxide flakes are added and the reactionmixture is cooled to −3° C. in an ice/acetone cooling bath. The solutionof the above-obtained homomorpholin-4-yl-acetaldehyde hydrochloride(staring compound II) is then added drop wise within 5 min at atemperature of 0° C. After the addition has ended the reaction mixtureis stirred for another 10 min at 0° C. and for a further hour at ambienttemperature. For working up 100 ml of ethyl acetate are added and theaqueous phase is separated off. The organic phase is washed withsaturated sodium chloride solution, dried over magnesium sulphate andevaporated down. The crude product is purified by chromatography over asilica gel column using ethyl acetate/methanol/conc. methanolic ammoniaas eluant. The product obtained is stirred with a little di-isopropylether, suction filtered and dried.

-   Yield: 2.40 g of (63% of theory)-   R_(f) value: 0.09 (silica gel, ethyl acetate/methanol/conc. aqueous    ammonia=90:10:1)-   Mass spectrum (ESI⁺): m/z=542, 544 [M+H]⁺

Inhibitors of the EGF Receptor and/or VEGF Receptor and/or IntegrinReceptors or any Other Protein Tyrosine Kinase Receptors, which are notClassified Under the Synthetic Small-Molecules

Inhibitors of the EGF receptor and/or VEGF receptor and/or integrinreceptors or any other protein tyrosine kinase receptors, which are notclassified under the synthetic small-molecules, which are of specialinterest, are the monoclonal antibodies directed to EGF receptor and/orVEGF receptor and/or integrin receptors or any other protein tyrosinekinase receptors. Representative compounds are, for example, atrasentan(integrin antagonist), rituximab, cetuximab, Avastin™ (bevacizumab),IMC-1C11, erbitux (C-225), DC-101, EMD-72000 (humanized EGFreceptor-specific monoclonal antibody), vitaxin (antibody directedagainst the α, β₃ integrin), and imatinib (c-Kit inhibitor). Monoclonalantibodies which can specifically recognize their antigen epitopes onthe relevant receptors, are in this respect of further special interest.The use of such antibodies, which were successful in vitro and in animalmodels, have not shown satisfying efficacy in patients as mono-drugtherapy. Similar results were obtained when other anti-angiogenic or EGFreceptor antagonists than antibodies were used in clinical trials. Itseems that tumours, if some specific sites are blocked, may use othercell surface molecules to compensate for said original blocking. Thus,tumours do not really shrink during various anti-angiogenic oranti-proliferative therapies. For these reasons, combination therapieswere in this case already proposed to circumvent this problem using, forexample, monoclonal antibodies together with specific cytotoxic orchemotherapeutic agents or in combination with radiotherapy orradio-immunotherapy. Indeed, clinical trials have shown that thesecombination therapies are more efficient than the correspondingmono-administrations.

Inhibitors Directed to EGF Receptor and/or VEGF Receptor and/or IntegrinReceptors or any Other Protein Tyrosine Kinase Receptors, which areFusion Proteins

A representative compound of this class is, for example, the compoundwith name VEGFtrap, developed by the pharmaceutical companies Regeneronand Aventis.

Compounds which Interact with Nucleic Acids and which are Classified asAlkylating Agents or Platinum Compounds

Compounds which interact with nucleic acids and which are classified asalkylating agents or platinum compounds, have already been described fortheir use for the treatment of diseases of oncological nature.Representative classes and examples of compounds are melphalan,cyclophosphamide, oxazaphosphorines, cisplatin, carboplatin,oxaliplatin, satraplatin, tetraplatin, iproplatin, mitomycin,streptozocin, carmustine (BCNU), lomustine (CCNU), busulfan, ifosfamide,streptozocin, thiotepa, chlorambucil, nitrogen mustards (such asmechlorethamine), ethyleneimine compounds and alkylsulphonates.

Compounds which Interact with Nucleic Acids and which are Classified asAnthracyclines, as DNA Intercalators or as DNA Cross-Linking Agents

Compounds which interact with nucleic acids and which are classified asanthracyclines, as DNA intercalators (including DNA minor-groove bindingcompounds) or as DNA cross-linking agents are also of interest for thetreatment of diseases of oncological nature. Representative classes andexamples of compounds are daunorubicin, doxorubicin (adriamycin),liposomal doxorubicin (doxil), epirubicin, idarubicin, mitoxantrone,amsacrine, dactinomycin, distamycin and derivatives, netropsin,pibenzimol, mitomycin, CC-1065 (Streptomyces zelensis fermentationproduct), duocarmycins, mithramycin, chromomycin, olivomycin,phtalanilides (propamidine, stilbamidine), anthramycins, aziridines ornitrosoureas and their derivatives.

Anti-Metabolites

Representative classes of anti-metabolites of interest are thepyrimidine and purine analogues or antagonists such as fluoropyrimidinesand thiopurines, or inhibitors of the nucleoside diphosphate reductase.Representative compounds are, for example, cytarabine, 5-fluorouracile(5-FU), uracil mustard, fludarabine, gemcitabine, capecitabine,mercaptopurine, cladribine, thioguanine, methotrexate, pentostatin,hydroxyurea, or folic acid.

Naturally Occurring, Semi-Synthetic or Synthetic Bleomycin TypeAntibiotics (BLM-Group Antibiotics)

Representative classes and compounds of interest are the phleomycins,bleomycins, bleomycin derivatives and salts, CHPP, BZPP, MTPP, BAPP,liblomycin. These agents are believed to mediate their therapeuticeffects via degradation of chromosomal DNA or RNA degradation(especially selective tRNA strand scission).

Inhibitors of DNA Transcribing Enzymes, Especially Topoisomerase I orTopoisomerase II Inhibitors

A representative class and examples of compounds of interest are theacridines and acridine derivatives, rifamycins, actinomycins, adramycin,camptothecins (irinotecan or camptosar, topotecan), amsacrines andanalogues, and the tricyclic carboxamides.

Chromatin Modifying Agents

A representative class of compounds of interest are thehistonedeacetylase inhibitors, such as SAHA (suberoylanilide hydroxamicacid), MD-275, trichostatin A, CBHA (M-carboxycinnamic acidbishydroxamide), LAQ824, or valproic acid.

Mitosis Inhibitors, Anti-Mitotic Agents, or Cell-Cycle Inhibitors

Representative classes and examples of compounds of interest are theanti-cancer drugs from plants, such as the taxanes (paclitaxel or taxol,docetaxel or taxotere), the vinca alkaloids (navelbine, vinblastin,vincristin, vindesine or vinorelbine), the tropolone alkaloids(colchicine and derivatives), the macrolides (maytansine, ansamitocins,rhizoxin), the antimitotic peptides (phomopsin, dolastatin), theepipodophyllotoxins or the derivatives of podophyllotoxin (etoposide,teniposide), the steganacins and the antimitotic carbamate derivatives(combretastatin, amphetinile), or procarbazine. These compounds are cdkinhibitors, tubulin binders or inhibitors of the polo-like kinase.

Proteasome Inhibitors

A representative compound of interest belonging to this class is, forexample, Velcade™ (bortezomib or PS-341).

Enzymes

Representative compounds and classes of interest are, for example,asparaginase, pegylated asparaginase (pegaspargase), and thethymidine-phosphorylase inhibitors.

Hormones, Hormone Antagonists or Hormone Inhibitors, or Inhibitors ofSteroid Biosynthesis

Representative classes and examples of hormones of interest are, forexample, the gestagens and estrogens, such as estramustine or T-66, ormegestrol. Representative classes and examples of hormone antagonists orinhibitors of interest are, for example, the anti-androgens, such asflutamide, casodex, anandron and cyproterone acetate, the aromataseinhibitors, such as amonogluthetimide, anastrozole, formestan andletrozole, the GNrH analogues, such as leuprorelin, buserelin, goserelinand triptorelin, the anti-estrogens, such as tamoxifen and especiallyits citrate salt, droloxifene, trioxifene, raloxifene, zindoxifene, thederivatives of 17β-estradiol (ICI 164,384 and ICI 182,780),aminoglutethimide, formestane, fadrozole, finasteride, or ketoconazole,or the LH-RH antagonist leuprolide. Steroid hormone inhibitors areespecially suitable for the treatment of breast and prostate cancer.

Steroids

Representative compounds of interest are, for example, prednisone,prednisolone, methylprednisolone, dexamethasone, budenoside,fluocortolone and triamcinolone. The reasons why steroids may be used inthe treatment of some cancers and the effects obtained with steroids inthe treatment of cancer depends on the type of cancer to be treated. Inthe treatment of solid tumors, steroids are in first line used tocontrol the symptoms. In the case of brain metastasis, they belong tothe standard therapy for reducing oedema. They are also used to controlthe inflammation which surrounds the tumor lesions. In the treatment ofhaematologic malignant neoplasias of lymphatic cell lines (ALL,non-Hodgkin lymphoma, myeloma), due to their cytolytic effect, steroidsare used as a real anti-tumor therapy, alone or in combination withclassical chemotherapeutic agents. The naturally occuring steroidtetrahydrocortisol, the synthetic cyclodextrin derivativeβ-cyclodextrine tetradecasulfate and the tetracycline derivativeminocycline, due to their antiangiogenic activity, have been suggestedfor a combination treatment with cytotoxic standard anticancertherapies, such as platinum, melphalan, cyclophosphamide, adriamycin,bleomycin or radiation based therapies (Teicher et al., Cancer research,Vol. 52, pp. 6702-6704, 1992). The steroid dexamethasone has also beentested as primary treatment of multiple myeloma (Dimopoulos et al.,Blood, Vol. 80(4), pp. 887-890, 1992).Furthermore, evaluation studies ofcombination therapies using dexamethasone and thalidomide, a substanceknown for its activity as TNF-α synthesis inhibitor and cytokineantagonist, have been disclosed recently. These studies focussed onpreviously untreated multiple myeloma (Weber et al., Journal of ClinicalOncology, Vol. 21, No. 1, pp. 16-19, 2003), newly diagnosed myeloma(Rajkumar et al., Journal of Clinical Oncology, Vol. 20, No. 21, pp.4319-4323, 2002) and multiple myeloma after intensive chemotherapy (Ann.Oncol., Vol. 13, No. 7, pp. 1116-1119, 2002).

With regard to all aspects of the invention, suitable steroids for thecombination treatment are meant to include in a non-limiting mannerprednisone, prednisolone, methylprednisolone, dexamethasone, budenoside,fluocortolone and triamcinolone. The preferred steroid is dexamethasone.

Cytokines, Hypoxia-Selective Cytotoxins, Inhibitors of Cytokines,Lymphokines, Antibodies Directed Against Cytokines or Oral andParenteral Tolerance Induction Agents

Representative classes and examples of compounds of interest areinterferons (especially interferon β), interleukins (especially IL-10and 12), anti-TNFα antibodies (etanercept), Immunomodulatory drugs (orIMiDs, especially inhibitors of the TNF-α production, such asthalidomide, its R- and S-enantiomers and its derivatives, or revimid(CC-5013)), leukotrien antagonists, mitomycin C, aziridoquinones(BMY-42355, AZQ, EO-9), 2-nitroimidazoles (misonidazole, NLP-1, NLA-1),nitroacridines, nitroquinolines, nitropyrazoloacridines, “dual-function”nitro aromatics (RSU-1069, RB-6145), nitro aromatic deactivated mustards(CB-1954), N-oxides of nitrogen mustards (nitromin), metal complexes ofnitrogen mustards, anti-CD3 or anti-CD25 antibodies, geneticallymodified enteric bacteria to achieve tolerance.

Supportive Agents

A representative class of compounds of interest are, for example, thebiphosphonates and their derivatives, such as, for example, minodronicacid (YM-529, Ono-5920, YH-529), zoledronic acid monohydrate,ibandronate sodium hydrate, clodronate disodium. These compounds are inclinical development or have been recently approved for the treatment ofbone metastasis from breast/lung cancer and for the treatment ofmultiple myeloma (Drugs of the Future 2002, 27(10), pp. 935-941).

Chemical Radiation Sensitizers and Protectors

Representative classes and compounds of interest are, for example, thenitroimidazoles (metronidazole, misonidazole, benznidazole, nimorazole)and further nitroaryl compounds such as RSU-1069, the nitroxyl andN-oxides such as SR-4233, the halogenated pyrimidine analogues(bromodeoxyuridine, iododeoxyuridine), or the thiophosphates (forexample WR-2721) as radiation protectors.

Photochemically Activated Drugs

Representative classes and compounds of interest are, for example,porfimer, photofrin, the benzoporphyrin derivatives, the pheophorbidederivatives, merocyanin 540 (MC-540), and tin etioporpurin.

Synthetic Poly- or Oligonucleotides

Synthetic poly- or oligonucleotides, which may optionally be modified orconjugated are also of interest. Representative classes of poly- oroligonucleotides are, for example, anti-templates RNAs and DNAs(synthetic or chemically modified oligonucleotides which are inactiveper se but capable of competing with functional template-primers fortheir specific binding site on an enzyme and thereby blocking theirfunctions), anti-sense RNAs and DNAs (sequence-specific inhibitors ofprotein synthesis which hybridize with complementary base sequences of agiven m-RNA, such as oblimersen), especially directed againstonco-genes, growth factor genes or tumor suppressor genes, antigenepoly- or oligonucleotides (oligonucleotides capable of forming triplexDNA structures which selectively inhibit the transcription of a targetgene), and ribozymes.

Non-Steroidal Anti-Inflammatory Drugs

Non-steroidal inflammatory drugs (NSAIDs) represent also an interestingclass of compounds which may be used for a combination therapy withinthe meaning of the present invention. Cyclo-oxygenase (COX) inhibitorsare of special interest, such as the non-selective COX inhibitorsacetylsalicyclic acid, mesalazin, ibuprofen, naproxen, flurbiprofen,fenoprofen, fenbufen, ketoprofen, indoprofen, pirprofen, carprofen,oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen,tiaprofenic acid, fluprofen, indomethacin, sulindac, tolmetin,zomepirac, nabumetone, diclofenac, fenclofenac, alclofenac, bromfenac,ibufenac, aceclofenac, acemetacin, fentiazac, clidanac, etodolac,oxpinac, mefenamic acid, meclofenamic acid, flufenamic acid, nifluminicacid, tolfenamic acid, diflunisal, flufenisal, piroxicam, tenoxicam,lornoxicam and nimesulide or the pharmaceutically acceptable saltsthereof, or the selective COX inhibitors meloxicam, celecoxib orrofecoxib. The selective COX-2 inhibitor meloxicam is especiallypreferred.

Other Chemotherapeutic or Naturally Occurring, Semi-Synthetic orSynthetic Therapeutic Agents

Further classes and examples of compounds are of interest for acombination therapy within the meaning of the present invention, suchas, for example, cytotoxic antibiotics, antibodies targeting surfacemolecules of cancer cells (especially HLA-DR antibodies such as, forexample, apolizumab and 1D09C3), inhibitors of metalloproteinases(TIMP-1, TIMP-2), Zinc, inhibitors of oncogenes (especially c-myc, Ras,v-raf or c-src inhibitors, such as P53 and Rb), inhibitors of genetranscription (especially the inhibitors of the transcription factorcomplex ESX/DRIP130/Sur-2 which controls the expression of Her-2, suchas those described in WO 03/097855) or of RNA translation or proteinexpression (especially the inhibitors of HER-2 expression, such as theheat shock protein HSP90 modulator geldanamycin and its derivative17-allylaminogeldanamycin or 17-AAG), complexes of rare earth elementssuch as the heterocyclic complexes of lanthanides described for examplein German Patent Nr. 101 38 538, photo-chemotherapeutic agents (PUVA, acombination of psoralen (P) and long-wave ultraviolet radiation (UVA)),IM-842, tetrathiomolybdate, squalamine, combrestatin A4, TNP-470,marimastat, neovastat, bicalutamide, abarelix, oregovomab, mitumomab,TLK-286, alemtuzumab, ibritumomab, temozolomide, denileukin diftitox,aldesleukin, dacarbazine, floxuridine, plicamycin, mitotane, pipobroman,plicamycin, tamloxifen, testolactone.

In a preferred embodiment in accordance with the present invention, thefurther chemotherapeutic or naturally occurring, semi-synthetic orsynthetic therapeutic agent is selected from synthetic small moleculeVEGF receptor antagonists, small molecule growth factor receptorantagonists, inhibitors of the EGF receptor and/or VEGF receptor and/orintegrin receptors or any other protein tyrosine kinase receptors whichare not classified under the synthetic small-molecules, inhibitorsdirected to EGF receptor and/or VEGF receptor and/or integrin receptorsor any other protein tyrosine kinase receptors, which are fusionproteins, compounds which interact with nucleic acids and which areclassified as alkylating agents or platinum compounds, compounds whichinteract with nucleic acids and which are classified as anthracyclines,as DNA intercalators or as DNA cross-linking agents, including DNAminor-groove binding compounds, anti-metabolites, naturally occurring,semi-synthetic or synthetic bleomycin type antibiotics, inhibitors ofDNA transcribing enzymes, and especially the topoisomerase I ortopoisomerase II inhibitors, chromatin modifying agents, mitosisinhibitors, anti-mitotic agents, cell-cycle inhibitors, proteasomeinhibitors, enzymes, hormones, hormone antagonists, hormone inhibitors,inhibitors of steroid biosynthesis, steroids, cytokines,hypoxia-selective cytotoxins, inhibitors of cytokines, lymphokines,antibodies directed against cytokines, oral and parenteral toleranceinduction agents, supportive agents, chemical radiation sensitizers andprotectors, photo-chemically activated drugs, synthetic poly- oroligonucleotides, optionally modified or conjugated, non-steroidalanti-inflammatory drugs, cytotoxic antibiotics, antibodies targetingsurface molecules of cancer cells, and especially the HLA-DR antibodiessuch as, inhibitors of metalloproteinases, metals, inhibitors ofoncogenes, inhibitors of gene transcription or of RNA translation orprotein expression, complexes of rare earth elements, orphoto-chemotherapeutic agents.

In a further preferred embodiment in accordance with the presentinvention, the further chemotherapeutic or naturally occurring,semi-synthetic or synthetic therapeutic agent is selected from a smallmolecule VEGF receptor antagonist such as vatalanib (PTK-787/ZK222584),SU-5416, SU-6668, SU-11248, SU-14813, AZD-6474, AZD-2171, CP-547632,CEP-7055, AG-013736, IM-842 or GW-786034, a dual EGFR/HER2 antagonistsuch as gefitinib, erlotinib, CI-1033 or GW-2016, an EGFR antagonistsuch as iressa (ZD-1839), tarceva (OSI-774), PKI-166, EKB-569, HKI-272or herceptin, an antagonist of the mitogen-activated protein kinase suchas BAY-43-9006 or BAY-57-9006, a quinazoline derivative such as4-[(3-chloro-4-fluorophenyl)amino]-6-{[4-(N,N-dimethylamino)-1-oxo-2-buten-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)-quinazolineor4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-(homomorpholin-4-yl)-1-oxo-2-buten-1-yl]amino}-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline,or a pharmaceutically acceptable salt thereof, a protein kinase receptorantagonist which is not classified under the synthetic small moleculessuch as atrasentan, rituximab, cetuximab, Avastin™ (bevacizumab),IMC-1C11, erbitux (C-225), DC-101, EMD-72000, vitaxin, imatinib, aprotein tyrosine kinase inhibitor which is a fusion protein such asVEGFtrap, an alkylating agent or a platinum compound such as melphalan,cyclophosphamide, an oxazaphosphorine, cisplatin, carboplatin,oxaliplatin, satraplatin, tetraplatin, iproplatin, mitomycin,streptozocin, carmustine (BCNU), lomustine (CCNU), busulfan, ifosfamide,streptozocin, thiotepa, chlorambucil, a nitrogen mustard such asmechlorethamine, an ethyleneimine compound, an alkylsulphonate,daunorubicin, doxorubicin (adriamycin), liposomal doxorubicin (doxil),epirubicin, idarubicin, mitoxantrone, amsacrine, dactinomycin,distamycin or a derivative thereof, netropsin, pibenzimol, mitomycin,CC-1065, a duocarmycin, mithramycin, chromomycin, olivomycin, aphtalanilide such as propamidine or stilbamidine, an anthramycin, anaziridine, a nitrosourea or a derivative thereof, a pyrimidine or purineanalogue or antagonist or an inhibitor of the nucleoside diphosphatereductase such as cytarabine, 5-fluorouracile (5-FU), uracil mustard,fludarabine, gemcitabine, capecitabine, mercaptopurine, cladribine,thioguanine, methotrexate, pentostatin, hydroxyurea, or folic acid, aphleomycin, a bleomycin or a derivative or salt thereof, CHPP, BZPP,MTPP, BAPP, liblomycin, an acridine or a derivative thereof, arifamycin, an actinomycin, adramycin, a camptothecin such as irinotecan(camptosar) or topotecan, an amsacrine or analogue thereof, a tricycliccarboxamide, an histonedeacetylase inhibitor such as SAHA, MD-275,trichostatin A, CBHA, LAQ824, or valproic acid, an anti-cancer drug fromplants such as paclitaxel (taxol), docetaxel or taxotere, a vincaalkaloid such as navelbine, vinblastin, vincristin, vindesine orvinorelbine, a tropolone alkaloid such as colchicine or a derivativethereof, a macrolide such as maytansine, an ansamitocin or rhizoxin, anantimitotic peptide such as phomopsin or dolastatin, anepipodophyllotoxin or a derivative of podophyllotoxin such as etoposideor teniposide, a steganacin, an antimitotic carbamate derivative such ascombretastatin or amphetinile, procarbazine, a proteasome inhibitor suchas Velcade™ (bortezomib or PS-341), an enzyme such as asparaginase,pegylated asparaginase (pegaspargase) or a thymidine-phosphorylaseinhibitor, a gestagen or an estrogen such as estramustine (T-66) ormegestrol, an anti-androgen such as flutamide, casodex, anandron orcyproterone acetate, an aromatase inhibitor such as aminogluthetimide,anastrozole, formestan or letrozole, a GNrH analogue such asleuprorelin, buserelin, goserelin or triptorelin, an anti-estrogen suchas tamoxifen or its citrate salt, droloxifene, trioxifene, raloxifene orzindoxifene, a derivative of 17β-estradiol such as ICI 164,384 or ICI182,780, aminoglutethimide, formestane, fadrozole, finasteride,ketoconazole, a LH-RH antagonist such as leuprolide, a steroid such asprednisone, prednisolone, methylprednisolone, dexamethasone, budenoside,fluocortolone or triamcinolone, an interferon such as interferon β, aninterleukin such as IL-10 or IL-12, an anti-TNFα antibody such asetanercept, an immunomodulatory drug such as thalidomide, its R- andS-enantiomers and its derivatives, or revimid (CC-5013), a leukotrienantagonist, mitomycin C, an aziridoquinone such as BMY-42355, AZQ orEO-9, a 2-nitroimidazole such as misonidazole, NLP-1 or NLA-1, anitroacridine, a nitroquinoline, a nitropyrazoloacridine, a“dual-function” nitro aromatic such as RSU-1069 or RB-6145, CB-1954, aN-oxide of nitrogen mustard such as nitromin, a metal complex of anitrogen mustard, an anti-CD3 or anti-CD25 antibody, a toleranceinduction agent, a biphosphonate or derivative thereof such asminodronic acid or its derivatives (YM-529, Ono-5920, YH-529),zoledronic acid monohydrate, ibandronate sodium hydrate or clodronatedisodium, a nitroimidazole such as metronidazole, misonidazole,benznidazole or nimorazole, a nitroaryl compound such as RSU-1069, anitroxyl or N-oxide such as SR-4233, an halogenated pyrimidine analoguesuch as bromodeoxyuridine, iododeoxyuridine, a thiophosphate such asWR-2721, a photo-chemically activated drug such as porfimer, photofrin,a benzoporphyrin derivative, a pheophorbide derivative, merocyanin 540(MC-540) or tin etioporpurin, an anti-template or an anti-sense RNA orDNA such as oblimersen, a non-steroidal inflammatory drug such asacetylsalicyclic acid, mesalazin, ibuprofen, naproxen, flurbiprofen,fenoprofen, fenbufen, ketoprofen, indoprofen, pirprofen, carprofen,oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen,tiaprofenic acid, fluprofen, indomethacin, sulindac, tolmetin,zomepirac, nabumetone, di-clofenac, fenclofenac, alclofenac, bromfenac,ibufenac, ace-clofenac, acemetacin, fentiazac, clidanac, etodolac,oxpinac, mefenamic acid, meclofenamic acid, flufenamic acid, nifluminicacid, tolfenamic acid, diflunisal, flufenisal, piroxicam, tenoxicam,lornoxicam, nimesulide, meloxicam, celecoxib, rofecoxib, or apharmaceutically acceptable salt of a non-steroidal inflammatory drug, acytotoxic antibiotic, an antibody targeting the surface molecules ofcancer cells such as apolizumab or 1D09C3, an inhibitor ofmetalloproteinases such as TIMP-1 or TIMP-2, Zinc, an inhibitor ofoncogenes such as P53 and Rb, a complex of rare earth elements such asthe heterocyclic complexes of lanthanides, a photo-chemotherapeuticagent such as PUVA, an inhibitor of the transcription factor complexESX/DRIP130/Sur-2, an inhibitor of HER-2 expression, such as the heatshock protein HSP90 modulator geldanamycin and its derivative17-allylaminogeldanamycin or 17-AAG, or a therapeutic agent selectedfrom IM-842, tetrathiomolybdate, squalamine, combrestatin A4, TNP-470,marimastat, neovastat, bicalutamide, abarelix, oregovomab, mitumomab,TLK-286, alemtuzumab, ibritumomab, temozolomide, denileukin diftitox,aldesleukin, dacarbazine, floxuridine, plicamycin, mitotane, pipobroman,plicamycin, tamloxifen or testolactone.

In a further preferred embodiment in accordance with the presentinvention, the further chemotherapeutic or naturally occurring,semi-synthetic or synthetic therapeutic agent is selected from ananti-cancer drug from plants such as paclitaxel (taxol), docetaxel ortaxotere, a vinca alkaloid such as navelbine, vinblastin, vincristin,vindesine or vinorelbine, a vinca alkaloid such as navelbine,vinblastin, vincristin, vindesine or vinorelbine, an alkylating agent ora platinum compound such as melphalan, cyclophosphamide, anoxazaphosphorine, cisplatin, carboplatin, oxaliplatin, satraplatin,tetraplatin, iproplatin, mitomycin, streptozocin, carmustine (BCNU),lomustine (CCNU), busulfan, ifosfamide, streptozocin, thiotepa,chlorambucil, a nitrogen mustard such as mechlorethamine, animmunomodulatory drug such as thalidomide, its R- and S-enantiomers andits derivatives, or revimid (CC-5013)), an ethyleneimine compound, analkylsulphonate, daunorubicin, doxorubicin (adriamycin), liposomaldoxorubicin (doxil), epirubicin, idarubicin, mitoxantrone, amsacrine,dactinomycin, distamycin or a derivative thereof, netropsin, pibenzimol,mitomycin, CC-1065, a duocarmycin, mithramycin, chromomycin, olivomycin,a phtalanilide such as propamidine or stilbamidine, an anthramycin, anaziridine, a nitrosourea or a derivative thereof, a pyrimidine or purineanalogue or antagonist or an inhibitor of the nucleoside diphosphatereductase such as cytarabine, 5-fluorouracile (5-FU), uracil mustard,fludarabine, gemcitabine, capecitabine, mercaptopurine, cladribine,thioguanine, methotrexate, pentostatin, hydroxyurea, or folic acid, anacridine or a derivative thereof, a rifamycin, an actinomycin,adramycin, a camptothecin such as irinotecan (camptosar) or topotecan,an amsacrine or analogue thereof, a tricyclic carboxamide, anhistonedeacetylase inhibitor such as SAHA, MD-275, trichostatin A, CBHA,LAQ824, or valproic acid, a proteasome inhibitor such as Velcade™(bortezomib or PS-341), a small molecule VEGF receptor antagonist suchas vatalanib (PTK-787/ZK222584), SU-5416, SU-6668, SU-11248, SU-14813,AZD-6474, AZD-2171, CP-547632, CEP-7055, AG-013736, IM-842 or GW-786034,an antagonist of the mitogen-activated protein kinase such asBAY-43-9006 or BAY-57-9006, a dual EGFR/HER2 antagonist such asgefitinib, erlotinib, CI-1033 or GW-2016, an EGFR antagonist such asiressa (ZD-1839), tarceva (OSI-774), PKI-166, EKB-569, HKI-272 orherceptin, a quinazoline derivative such as4-[(3-chloro-4-fluorophenyl)amino]-6-{[4-(N,N-dimethylamino)-1-oxo-2-buten-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)-quinazolineor4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-(homomorpholin-4-yl)-1-oxo-2-buten-1-yl]amino}-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline,or a pharmaceutically acceptable salt thereof, an inhibitor of thetranscription factor complex ESX/DRIP130/Sur-2, an inhibitor of HER-2expression, such as the heat shock protein HSP90 modulator geldanamycinand its derivative 17-allylaminogeldanamycin or 17-AAG, a protein kinasereceptor antagonist which is not classified under the synthetic smallmolecules such as atrasentan, rituximab, cetuximab, Avastin™(bevacizumab), IMC-1C11, erbitux (C-225), DC-101, EMD-72000, vitaxin,imatinib, or an antibody targeting the surface molecules of cancer cellssuch as apolizumab or 1D09C3.

In a further preferred embodiment in accordance with the presentinvention, the further chemotherapeutic or naturally occurring,semi-synthetic or synthetic therapeutic agent is selected from theabove-mentioned quinazoline derivative disclosed in WO 02/50043 asexemplified compound of Example 1(10), namely4-[(3-chloro-4-fluorophenyl)amino]-6-{[4-(N,N-dimethylamino)-1-oxo-2-buten-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)-quinazoline,or the tautomers, the stereoisomers and the salts thereof, particularlythe physiologically and pharmaceutically acceptable salts thereof withinorganic or organic acids or bases.

In a further preferred embodiment in accordance with the presentinvention, the further chemotherapeutic or naturally occurring,semi-synthetic or synthetic therapeutic agent is selected from thedi-maleic acid salt of the compound4-[(3-chloro-4-fluorophenyl)amino]-6-{[4-(N,N-dimethylamino)-1-oxo-2-buten-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)-quinazoline,or the tautomers or stereoisomers thereof.

In a further preferred embodiment in accordance with the presentinvention, the further chemotherapeutic or naturally occurring,semi-synthetic or synthetic therapeutic agent is selected from4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-(homomorpholin-4-yl)-1-oxo-2-buten-1-yl]amino}-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazolineor the physiologically and pharmaceutically acceptable salts thereofwith inorganic or organic acids or bases.

Radiation Therapy, Radio-Immunotherapy or Pre-TargetedRadioimmunotherapy

Radiation therapy, radio-immunotherapy or pre-targetedradioimmunotherapy are used for the treatment of diseases of oncologicalnature. “Radiotherapy”, or radiation therapy, means the treatment ofcancer and other diseases with ionizing radiation. Ionizing radiationdeposits energy that injures or destroys cells in the area being treated(the target tissue) by damaging their genetic material, making itimpossible for these cells to continue to grow. Radiotherapy may be usedto treat localized solid tumors, such as cancers of the skin, tongue,larynx, brain, breast, lung or uterine cervix. It can also be used totreat leukemia and lymphoma, i.e. cancers of the blood-forming cells andlymphatic system, respectively. One type of radiation therapy commonlyused involves photons, e.g. X-rays. Depending on the amount of energythey possess, the rays can be used to destroy cancer cells on thesurface of or deeper in the body. The higher the energy of the x-raybeam, the deeper the x-rays can go into the target tissue. Linearaccelerators and betatrons are machines that produce x-rays ofincreasingly greater energy. The use of machines to focus radiation(such as x-rays) on a cancer site is called external beam radiotherapy.Gamma rays are another form of photons used in radiotherapy. Gamma raysare produced spontaneously as certain elements (such as radium, uranium,and cobalt 60) release radiation as they decompose, or decay. Anothertechnique for delivering radiation to cancer cells is to placeradioactive implants directly in a tumor or body cavity. This is calledinternal radiotherapy. Brachytherapy, interstitial irradiation, andintracavitary irradiation are types of internal radiotherapy. In thistreatment, the radiation dose is concentrated in a small area, and thepatient stays in the hospital for a few days. Internal radiotherapy isfrequently used for cancers of the tongue, uterus, and cervix. A furthertechnique is intra-operative irradiation, in which a large dose ofexternal radiation is directed at the tumor and surrounding tissueduring surgery. Another approach is particle beam radiation therapy.This type of therapy differs from photon radiotherapy in that itinvolves the use of fast-moving subatomic particles to treat localizedcancers. Some particles (neutrons, pions, and heavy ions) deposit moreenergy along the path they take through tissue than do x-rays or gammarays, thus causing more damage to the cells they hit. This type ofradiation is often referred to as high linear energy transfer (high LET)radiation. Radio-sensitizers make the tumour cells more likely to bedamaged, and radio-protectors protect normal tissues from the effects ofradiation. Hyperthermia, the use of heat, may also be used forsensitizing tissue to radiation. Another option involves the use ofradio-labeled antibodies to deliver doses of radiation directly to thecancer site (radio-immunotherapy). There are numerous methods availablein the art to link a radioisotope to an antibody. For example, for theradio-iodination of the antibody, a method as disclosed in WO 93/05804may be employed. Another option is to use a linker molecule between theantibody and the radioisotope, e.g. MAG-3 (U.S. Pat. No. 5,082,930, EP 0247 866), MAG-2 GABA (U.S. Pat. No. 5,681,927, EP 0 284 071), and N2S2(phenthioate, U.S. Pat. No. 4,897,255, U.S. Pat. No. 5,242,679, EP 0 188256). A further option is pre-targeted radio-immunotherapy, which may beused to minimize the radiation toxicity by separating thelong-circulating antibody and the rapidly cleared radionuclide (Drugs ofthe future 2003, 28(2), pp. 167-173). Detailed protocols forradiotherapy are readily available to the expert (Cancer Radiotherapy:Methods and Protocols (Methods in Molecular Medicine), Huddart R A Ed.,Human Press 2002). The expert knows how to determine an appropriatedosing and application schedule, depending on the nature of the diseaseand the constitution of the patient. In particular, the expert knows howto assess dose-limiting toxicity (DLT) and how to determine the maximumtolerated dose (MTD) accordingly.

Co-Administration and/or Co-Treatment Therapies

Co-administration of the selected protein tyrosine kinase receptorantagonist and of the further chemotherapeutic or naturally occurring,semi-synthetic or synthetic therapeutic agent, and/or co-treatment withradiotherapy or radio-immunotherapy, is meant to include administrationand/or treatment sequential in time or si-multaneous administrationand/or treatment. For sequential administration and/or treatment, theselected protein tyrosine kinase receptor antagonist can be administeredbefore or after administration of the further chemotherapeutic ornaturally occurring, semi-synthetic or synthetic therapeutic agent,and/or before or after treatment with radiotherapy orradio-immunotherapy.

The active compounds can be administered orally, bucally, parenterally,by inhalation spray, rectally or topically, the oral administrationbeing preferred. Parenteral administration may include subcutaneous,intravenous, intramuscular and intrasternal injections and infusiontechniques.

The active compounds can be orally administered in a wide variety ofdifferent dosage forms, i.e., they may be formulated with variouspharmaceutically acceptable inert carriers in the form of tablets,capsules, lozenges, troches, hard candies, powders, sprays, aqueoussuspensions, elixirs, syrups, and the like. Such carriers include soliddiluents or fillers, sterile aqueous media and various non-toxic organicsolvents. Moreover, such oral pharmaceutical formulations can besuitably sweetened and/or flavoured by means of various agents of thetype commonly employed for such purposes. In general, the compounds ofthis invention are present in such oral dosage forms at concentrationlevels ranging from about 0.5% to about 90% by weight of the totalcomposition, in amounts which are sufficient to provide the desired unitdosages. Other suitable dosage forms for the compounds of this inventioninclude controlled release formulations and devices well known to thosewho practice in the art.

For purposes of oral administration, tablets containing variousexcipients such as sodium citrate, calcium carbonate and calciumphosphate may be employed along with various disintegrants such asstarch and preferably potato or tapioca starch, alginic acid and certaincomplex silicate, together with binding agents such aspolyvinylpyrrolidone, sucrose, gelatine and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc or compositions of a similar type may also be employed as fillersin soft and hard-filled gelatine capsules; included lactose or milksugar as well as high molecular weight polyethylene glycols. Whenaqueous suspensions and/or elixirs are desired for oral administration,the essential active ingredient therein may be combined with varioussweetening or flavouring agents, colouring matter or dyes and, if sodesired, emulsifying agents and/or water, ethanol, propylene glycol,glycerine and various like combinations thereof.

For purposes of oral administration, an especially suitablepharmaceutical formulation for the selected protein kinase receptorantagonist in accordance with the present invention is soft gelatinecapsules. Suitable soft gelatine capsules for the encapsulation ofpharmaceutical compounds and the process for their preparation aredescribed, for example, in GB patent No. 395546, U.S. Pat. No.2,720,463, U.S. Pat. No. 2,870,062, U.S. Pat. No. 4,829,057, and in thefollowing publications: ANON (Verpack-Rundsch., Vol. 21, No. 1, January1970, pp. 136-138), Lachman et al. (The Theory and Practice ofIndustrial Pharmacy, Chap. 13, published by Lea & Febiger, 1970), Ebert(Soft Gelatine Capsules: A Unique Dosage Form, reprint fromPharmaceutical Technology, October 1977) and R. F. Jimerson (SoftGelatine Capsule Update, Drug Development and Industrial Pharmacy, Vol.12 (8 & 9), pp. 1133-1144, 1986).

For purposes of parenteral administration, solutions of the compounds insesame or peanut oil or in aqueous propylene glycol may be employed, aswell as sterile aqueous solutions of the corresponding pharmaceuticallyacceptable salts. Such aqueous solutions should be suitably buffered ifnecessary, and the liquid diluent rendered isotonic with sufficientsaline or glucose. These particular aqueous solutions are especiallysuitable for intravenous, intramuscular and subcutaneous injectionpurposes. In this connection, the sterile aqueous media employed arereadily obtained by standard techniques well known to those skilled inthe art. For instance, distilled water is ordinarily used as the liquiddiluent and the final preparation is passed through a suitable bacterialfilter such as a sintered glass filter or a diatomaceous earth orunglazed porcelain filter. Preferred filters of this type include theBerkefeld, the Chamberland and the Asbestos Disk-Metal Seitz filter,wherein the fluid is sucked into a sterile container with the aid of asuction pump. The necessary steps should be taken throughout thepreparation of these inject-able solutions to insure that the finalproducts are obtained in a sterile condition.

For purposes of transdermal administration, the dosage form of theparticular compound or compounds may include, by way of example,solutions, lotions, ointments, creams, gels, suppositories,rate-limiting sustained release formulations and devices therefore. Suchdosage forms comprise the particular compound or compounds and mayinclude ethanol, water, penetration enhancer and inert carriers such asgel-producing materials, mineral oil, emulsifying agents, benzyl alcoholand the like.

In accordance with one embodiment, the selected protein tyrosine kinasereceptor antagonist, or its polymorph or pharmaceutically acceptablesalt, may be administered in a daily dosage such that the plasma levelof the active substance lies between 10 and 500 ng/ml for at least 12hours of a 24 hours dosing interval.

In accordance with a further embodiment, the selected protein tyrosinekinase receptor antagonist, or its polymorph or pharmaceuticallyacceptable salt, may be administered in a daily dosage of between 2 mgand 20 mg/kg body weight.

The further chemotherapeutic or naturally occurring, semi-synthetic orsynthetic therapeutic agent may be administered using suitable dosageforms, dosage levels and devices well known to those who practice in theart. In accordance with one embodiment, if the further chemotherapeuticor naturally occurring, semi-synthetic or synthetic therapeutic agent isa steroid, the steroid may be administered in a daily dosage of 5 to 500mg.

As already mentioned hereinbefore, detailed protocols for radiotherapyare readily available to the expert. The expert knows how to determinean appropriate dosing and application schedule, depending on the natureof the disease and the constitution of the patient. In particular, theexpert knows how to assess dose-limiting toxicity (DLT) and how todetermine the maximum tolerated dose (MTD) accordingly.

In Vitro and In Vivo Combination Studies Showing the Potency to Inhibitthe Proliferation and/or to Induce the Apoptosis of Tumour Cells

In the following examples of combinations, in vitro experiments withrepresentative cell lines or in vivo experiments with nude mice carryingspecific tumours, illustrate the potency of the combination of aselected protein tyrosine kinase antagonist with a furtherchemotherapeutic agent and/or with radiotherapy to inhibit theproliferation of endothelial or tumour cells and/or to induce theapoptosis of tumour cells. These examples are thus illustrative of thepresent invention.

Examples of Combinations

-   -   1. Combination of an antagonist of at least one receptor        selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR,        HER2, IGF1R, HGFR or c-Kit, which is further an antagonist of a        src tyrosine kinase family member, or a polymorph, metabolite or        pharmaceutically acceptable salt thereof, and of a steroid, for        the treatment of refractory or relapsed multiple myeloma

In vitro studies performed with the monoethanesulfonate salt of3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone(compound MES(T)) have shown that this specific compound has unexpectedproperties which makes it especially suitable for the treatment of thediseases in accordance with the present invention, especially whencombined with a steroid, and more specifically with dexamethasone.

Amongst these unexpected properties, the following are of particularrelevance for the target indications: Tyrosine kinase inhibition ofVEGFR1 to 3, FGFR1 and 3, PDGFR α; Inhibition of src-tyrosine kinasefamily members and potential inhibition of the proliferation of myelomacells; Inhibition of the neo-angiogenesis induced by VEGF and bFGF;Inhibition of the paracrine IL-6 secretion; Inhibition of the cellcontact mediated IL-6 secretion; Inhibition of the autocrine VEGF andbFGF effects; Direct induction of apoptosis on cell lines with t(4;14).

This specific compound appears to be further especially suitable for thetreatment of multiple myeloma. The following recent findings constitutea line of evidence for the selection of this specific compound for thisindication: Neovascularization parallels infiltration of bone marrow ina murine multiple myeloma model (Yaccoby et al., Blood 1998, Vol. 92(8),pp. 2908-2913) and in multiple myeloma patients undergoing progression(Vacca et al., Blood 1999, Vol. 93(9), pp. 3064-3073; Kumar et al.,Blood 2002, Blood First Edition Paper, Pre-published Online Oct. 17,2002, DOI 10.1182/blood-2002-08-2441); VEGF has been shown to be apotent stimulus of angiogenesis (Toi et al., Lancet Oncol. 2001, Vol. 2,pp. 667-673); VEGF is expressed in and secreted by multiple myelomacells (Dankbar et al., Blood 2000, Vol. 95(8), pp. 2630-2636; Bellamy etal., Cancer Res. 1999, Vol. 59(3), pp. 728-33); VEGF induces IL-6secretion from marrow stromal cells, which in turn augments VEGFexpression from clonal plasma cells (Dankbar et al., Blood 2000, Vol.95(8), pp. 2630-2636); IL-6 is considered a major growth factor formultiple myeloma cells in vivo (Klein et al., Blood 1995, Vol. 85(4),pp. 863-872); IL-6 inhibits Dexamethasone-induced myeloma cell death(Hardin et al., Blood 1994, Vol. 84(9), pp. 3063-3070); VEGF inducesproliferation and triggers migration of multiple myeloma cells (Podar etal., Blood 2001, Vol. 98(2), pp. 428-435); VEGF enhances osteoclasticbone resorption, which is a characteristic feature of multiple myeloma(Nakagawa et al., FEBS Lett. 2000, Vol. 473(2), pp. 161-164; Niida etal., J. Exp. Med. 1999, Vol. 190(2), pp. 293-298); FGFR3 inducesproliferation, inhibits apoptosis and is involved in progression ofmyeloma cells (Chesi et al., Blood 2001, Vol. 97(3), pp. 729-736;Plowright et al., Blood 2000, Vol. 95(3), pp. 992-998); FGFR3 isdysregulated and constitutively activated in a subset of myelomapatients (Chesi et al., Blood 2001, Vol. 97(3), pp. 729-736; Chesi etal., Nat. Genet. 1997, Vol. 16(3), pp. 260-264); Src family kinases areinvolved in proliferative responses induced in myeloma (Ishikawa et al.;Blood 2002, Vol. 99(6), pp. 2172-2178).

The following results of in vitro experiments evidence that theproperties of the compound MES(T) make it especially suitable for thetreatment of multiple myeloma.

In the first experiment, the inhibition effect of the compound MES(T) onthe secretion of IL-6 by bone marrow stromal cells (BMSC cells) wasinvestigated, at different concentrations (0, 10, 50, 125, 250 and 500nM) of MES(T), in native conditions (native) and in conditions ofstimulation of the cells with the bFGF (+ bFGF) or with the VEGF (+VEGF) growth factors. For comparison, the inhibition effect withinhibition of anti-bFGF (+ anti-bFGF), anti-VEGF (+ anti-VEGF) and acombination of anti-bFGF and anti-VEGF (+ anti-VEGF+anti-bFGF) were alsoinvestigated. The results of the experiment are shown in the followingTable II.

TABLE II MES (T) con- Inhibition of IL-6 secretion by BMSC cells cen-+anti- +anti- +anti-VEGF tration native +bFGF +VEGF bFGF VEGF +anti-bFGF 0 nM 124.2 216.9 107.4 77.7 118.9 71.1  10 nM 130.2 150.5 122.3 68.9148.6 68.1  50 nM 170.4 179.7 130.7 81.3 155.2 63.4 125 nM 97.5 91.2141.0 42.4 166.7 86.1 250 nM 76.5 76.9 65.5 33.0 89.4 45.0 500 nM 39.643.4 14.8 20.2 16.2 13.5

The results of this experiment show that the compound MES(T) atconcentration of ≧250 nM inhibits basal (native) as well asbFGF/VEGF-stimulated IL-6 secretion of bone marrow stromal cells (BMSCcells), and that the inhibition is more potent than the inhibitionobtained with the antibodies. Since the bFGF and VEGF growth factors(released by myeloma cells) have been previously shown to stimulate BMSCcells and the microvascular endothelium to produce and secrete IL-6,which itself stimulates myeloma cells to produce both the bFGF and VEGFgrowth factors, an inhibition of IL-6 secretion by the compound inaccordance with the present invention shows its potency for thetreatment of multiple myeloma.

In a further experiment, the inhibition effect of the compound MES(T) onthe secretion of IL-6 in transwell and contact co-cultures of myelomacells (U-266 myeloma cell lines) and bone marrow stromal cells (BMSCcells) was investigated, at different concentrations (0, 50, 125, 250and 500 nM) of MES(T). For comparison, the inhibition effect on BMSCmono-cultures (native) and, as control, the level of secretion of U266mono-cultures, were also investigated. The results of the experiment areshown in the following Table III.

TABLE III Inhibition of IL-6 secretion BMSC Transwell Contact U266MES(T) mono- U-266 + BMSC U-266 + BMSC mono- concentration culturesco-cultures co-cultures cultures  0 nM 153.5 336.1 348.1 2.0  50 nM213.4 354.5 125 nM 192.1 297.6 259.6 250 nM 69.9 231.1 199.4 500 nM 38.6123.9 114.7

The results of this experiment show that the compound MES(T) is able todecrease to its basal (native) value the level of IL-6 secretion of BMSCcultures stimulated by myeloma cells in transwell and contactco-cultures. Thus, it can be concluded that the compound MES(T)interferes with the myeloma-stroma interaction targeting the bone marrowmicroenvironment by significantly diminishing NFκB-dependent IL-6production. This further shows the potency of the compound in accordancewith the present invention for the treatment of multiple myeloma.

In further experiments, it could be shown that the compound MES(T)provides pro-apoptotic effects in t(14;16) MM1.s myeloma cells (MM1.smyeloma cells carrying the translocation t(14;16)), and that thecompound MES(T) enhances the apoptosis induced by dexamethasone.

Due to these properties, it can be concluded that the compound MES(T)isespecially suitable for a combination treatment of refractory orrelapsed multiple myeloma with a steroid, and especially dexamethasone.

-   -   2. Combination of an antagonist of at least one receptor        selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR,        HER2, IGF1R, HGFR or c-Kit, which is further an antagonist of a        src tyrosine kinase family member, or a polymorph, metabolite or        pharmaceutically acceptable salt thereof, and of a dual        antagonist of the epidermal growth factor (EGF) receptor and of        the human epidermal growth factor of type 2 (HE type 2)        receptor, for the treatment of prostate cancer, non-small cell        lung cancer or colorectal cancer

The following experiment was performed in order to investigate theeffect of a combination therapy with suboptimal doses of an antagonistof at least one receptor selected from VEGFR 1 to 3, PDGFRα and β,FGFR1, 2 and 3, EGFR, HER2, IGF1R, HGFR or c-Kit, which is further anantagonist of a src tyrosine kinase family member, namely thedi-chloride salt of(Z)-3-(1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-phenylamino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone(compound referred to as C12(T)), which is the di-chloride salt of aboveexemplified compound (T), and a dual antagonist of the epidermal growthfactor (EGF) receptor and of the human epidermal growth factor of type 2(HE type 2) receptor, namely the compound4-[(3-chloro-4-fluorophenyl)amino]-6-{[4-(N,N-dimethylamino)-1-oxo-2-buten-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)-quinazoline,(compound referred to as EGFR/HER2 inh., and described in WO 02/50043 asexemplified compound of Example 1(10)), on the reduction of tumourgrowth, in comparison to the mono-therapies at the same doses.

For this purpose, nude mice (NMRI nu/nu) were injected subcutaneouslywith SKOV-3 cells (human ovarian carcinoma). Mice carrying establishedtumours were randomised into control and treatment groups (N=10). Themice in the control group only received the carrier solution (0.5%Natrosol), the second group was treated daily per os with 15 mg/kgEGFR/HER2 inh., the third received once daily 50 mg/kg C12(T), and thefourth group of mice was treated with the combination of 15 mg/kgEGFR/HER2 inh. and 50 mg/kg C12(T). FIG. 3 shows the results of theexperiment.

Daily per os treatment was initially performed for 31 days. At this timepoint some of the mice from the control group carried tumours biggerthan 2000 mm³ and therefore had to be sacrificed. The calculated treatedtumour to control tumour (T/C) ratio at this time point was 35% for thegroup treated with 15 mg/kg EGFR/HER2 inh., 32% for the group treatedwith 50 mg/kg C12(T), and 13% for the group treated with thecombination. This result clearly demonstrates the anti-tumour effect ofthe combination of a VEGFR-2 and an EGFR/HER-2 inhibitor in vivo.Furthermore, continuing the treatment until day 64 shows extremely slowtumour growth in the combination group in comparison to the singletreatment group where the tumours eventually are growing to comparablesizes as the control treated tumours.

From the results of this experiment, it can thus be concluded that thecombination of compounds targeting different mechanisms involved in andimportant for tumour growth such as the VEGFR-2 inhibitor C12(T),inhibiting tumour angiogenesis, and the combined EGFR/HER-2 inhibitorEGFR/HER2 inh., inhibiting the proliferative signalling through theclass I receptor tyrosine kinases, have a synergistic anti-tumourefficacy. Thus, all combinations of inhibitors of tumour angiogenesis(e.g. the indolinone derivatives described in WO 02/36564, WO 99/52869,WO 00/18734, WO 00/73297, WO 01/27080, WO 01/27081 or WO 01/32651, thesmall molecule VEGF receptor antagonists described in WO 01/60814, WO99/48868, WO 98/35958, and especially the compounds vatalanib(PTK-787/ZK222584), SU-5416, SU-6668, SU-11248, SU-14813, AZD-6474,AZD-2171, CP-547632, CEP-7055, AG-013736, IM-842 or GW-786034, themonoclonal antibodies directed to the VEGF receptor, and especiallyAvastin™ (bevacizumab)or IMC-1C11) with EGFR inhibitors (e.g. iressa(ZD-1839), tarceva (OSI-774), PKI-166, EKB-569, HKI-272 or herceptin) orcombined EGFR/HER-2 inhibitors (e.g. the quinazoline derivativesdisclosed in WO 00/78735 and WO 02/50043, gefitinib, erlotinib, CI-1033or GW-2016) will expectedly have the same or similar effects foranti-tumour therapies.

-   -   3. Combination treatment of an antagonist of at least one        receptor selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and        3, EGFR, HER2, IGF1R, HGFR or c-Kit, which is further an        antagonist of a src tyrosine kinase family member, or a        polymorph, metabolite or pharmaceutically acceptable salt        thereof (e.g. the compound MES(T)), and of radiation therapy for        the treatment of breast cancer or ovarian cancer    -   4. Combination of an antagonist of at least one receptor        selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR,        HER2, IGF1R, HGFR or c-Kit, which is further an antagonist of a        src tyrosine kinase family member, or a polymorph, metabolite or        pharmaceutically acceptable salt thereof (e.g. the compound        MES(T)), and of a further antagonist of VEGFR 2, PDGFR or bFGFR        (e.g. vatalanib (PTK-787, ZD-6474, or the monoclonal antibody        Avastin™) or an antagonist of EGFR (e.g. tarceva (OSI-774)), for        the treatment of colorectal cancer, solid tumours, breast        cancer, non-small cell lung cancer, small cell lung cancer or        multiple myeloma    -   5. Combination of an antagonist of at least one receptor        selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR,        HER2, IGF1R, HGFR or c-Kit, which is further an antagonist of a        src tyrosine kinase family member, or a polymorph, metabolite or        pharmaceutically acceptable salt thereof (e.g. the compound        MES(T)), and of an antimetabolite (e.g. gemcitabine) and a        platinum compound (e.g. cisplatin), or of an anticancer drug        from plants (e.g. paclitaxel) and a platinum compound (e.g.        carboplatin), for the treatment of non-small cell lung cancer or        ovarian carcinoma    -   6. Combination of an antagonist of at least one receptor        selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR,        HER2, IGF1R, HGFR or c-Kit, which is further an antagonist of a        src tyrosine kinase family member, or a polymorph, metabolite or        pharmaceutically acceptable salt thereof (e.g. the compound        MES(T)), and of hormone antagonists (e.g. leuprorelin and        flutamide), for a continuous and/or intermittent treatment of        metastatic hormone sensitive prostate cancer    -   7. Combination of an antagonist of at least one receptor        selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR,        HER2, IGF1R, HGFR or c-Kit, which is further an antagonist of a        src tyrosine kinase family member, or a polymorph, metabolite or        pharmaceutically acceptable salt thereof (e.g. the compound        MES(T)), and of a derivative of podophyllotoxin (e.g. etoposide)        and a platinum compound (e.g. carboplatin or cisplatin), for the        treatment of small cell lung cancer    -   8. Combination of an antagonist of at least one receptor        selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR,        HER2, IGF1R, HGFR or c-Kit, which is further an antagonist of a        src tyrosine kinase family member, or a polymorph, metabolite or        pharmaceutically acceptable salt thereof (e.g. the compound        MES(T)), and of an anticancer drug from plants (e.g. paclitaxel        or taxol), for the treatment of ovarian carcinoma, small cell        lung cancer or prostate cancer    -   9. Combination of an antagonist of at least one receptor        selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR,        HER2, IGF1R, HGFR or c-Kit, which is further an antagonist of a        src tyrosine kinase family member, or a polymorph, metabolite or        pharmaceutically acceptable salt thereof (e.g. the compound        MES(T)), and of an anticancer drug from plants (e.g. taxotere)        for the treatment of prostate cancer    -   10. Combination of an antagonist of at least one receptor        selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR,        HER2, IGF1R, HGFR or c-Kit, which is further an antagonist of a        src tyrosine kinase family member, or a polymorph, metabolite or        pharmaceutically acceptable salt thereof (e.g. the compound        MES(T)), and of a platinum compound (e.g. carboplatin) and an        anticancer drug from plants (e.g. paclitaxel), for the treatment        of ovarian carcinoma, especially after debulking surgery    -   11. Combination of an antagonist of at least one receptor        selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR,        HER2, IGF1R, HGFR or c-Kit, which is further an antagonist of a        src tyrosine kinase family member, or a polymorph, metabolite or        pharmaceutically acceptable salt thereof (e.g. the compound        MES(T)), and of a topoisomerase I inhibitor (e.g. topotecan) and        an anthracycline (e.g. doxorubicin), for the treatment of        ovarian cancer    -   12. Combination of an antagonist of at least one receptor        selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR,        HER2, IGF1R, HGFR or c-Kit, which is further an antagonist of a        src tyrosine kinase family member, or a polymorph, metabolite or        pharmaceutically acceptable salt thereof (e.g. the compound        MES(T)), and of a topoisomerase I inhibitor (e.g. topotecan),        for the treatment of small cell lung cancer or ovarian carcinoma    -   13. Combination of an antagonist of at least one receptor        selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR,        HER2, IGF1R, HGFR or c-Kit, which is further an antagonist of a        src tyrosine kinase family member, or a polymorph, metabolite or        pharmaceutically acceptable salt thereof (e.g. the compound        MES(T)), and of an anticancer drug from plants (e.g. docetaxel)        and a steroid hormone (e.g. estramustine), for the treatment of        hormone refractory prostate cancer    -   14. Combination of an antagonist of at least one receptor        selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR,        HER2, IGF1R, HGFR or c-Kit, which is further an antagonist of a        src tyrosine kinase family member, or a polymorph, metabolite or        pharmaceutically acceptable salt thereof (e.g. the compound        MES(T)), and of a vinca alkaloid (e.g. navelbine) for the        treatment of lung cancer    -   15. Combination of an antagonist of at least one receptor        selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR,        HER2, IGF1R, HGFR or c-Kit, which is further an antagonist of a        src tyrosine kinase family member, or a polymorph, metabolite or        pharmaceutically acceptable salt thereof (e.g. the compound        MES(T)), and of a platinum compound (e.g. carboplatin or        cis-platin, preferably carboplatin) for the treatment of ovarian        carcinoma or non-small cell lung cancer    -   16. Combination of an antagonist of at least one receptor        selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR,        HER2, IGF1R, HGFR or c-Kit, which is further an antagonist of a        src tyrosine kinase family member, or a polymorph, metabolite or        pharmaceutically acceptable salt thereof (e.g. the compound        MES(T)), and of a COX-2 inhibitor (e.g. celecoxib, rofecoxib or        meloxicam), for the treatment of colon or rectal cancer    -   17. Combination of an antagonist of at least one receptor        selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR,        HER2, IGF1R, HGFR or c-Kit, which is further an antagonist of a        src tyrosine kinase family member, or a polymorph, metabolite or        pharmaceutically acceptable salt thereof (e.g. the compound        MES(T)), and of a 5-alpha reductase inhibitor (e.g.        finasteride), for the treatment of prostate cancer    -   18. Combination of an antagonist of at least one receptor        selected from VEGFR 1 to 3, PDGFRα and β, FGFR1, 2 and 3, EGFR,        HER2, IGF1R, HGFR or c-Kit, which is further an antagonist of a        src tyrosine kinase family member, or a polymorph, metabolite or        pharmaceutically acceptable salt thereof (e.g. the compound        MES(T)), and of a photo-chemotherapeutic agent (PUVA, a        combination of psoralen (P) and long-wave ultraviolet radiation        (UVA)), for the treatment of psoriasis

Essentially, for the treatment of oncological diseases, the rationalefor the combination treatment in accordance with the present inventionis that there is a therapeutic advantage for the cancer patient tocombine specific and mechanistically acting molecules with more broadlyacting therapeutic concepts in the following ways:

-   -   Through the combination the target cells will have less chance        to survive through possible escape mechanisms;    -   When compared to the doses used in a mono-therapy, due to an        additive or synergistic effect of the combination, the required        respective doses of the drugs can be reduced;    -   Scheduling of the respective drugs in a combination reduces the        likelihood of the tumour cells to develop resistances against        the drugs, leads to a better delivery of certain drugs to the        tumour (reduction of intratumoral pressure) and may activate        further death pathways in the tumour cells.

Thus, by targeting different cellular structures and compartments, thecombination therapies in accordance with the present invention areexpected to provide a clinically relevant benefit in survival or time totumour progression for larger patient population as the correspondingmono-therapies. As a result of the specific anti-angiogenic therapywith, for example, the compound MES(T), tumours seem to be less capableof recovering from the damage caused by conventional chemotherapy. Also,by blocking the effects of VEGF on vascular permeability, a decline ofthe interstitial pressure in tumours seems to occur, allowing a greaterpenetration of the cytotoxic drugs. Maintenance therapy with a specificanti-angiogenic agent such as, for example, the compound MES(T), afterstandard cytoreduction, seems also to result in a consolidation of theresponse obtained with the cytotoxic therapy. This approach issubstantiated by preclinical evidence that combinations ofanti-angiogenic compounds with cytotoxic therapies result in synergisticanti-tumour activity.

For the treatment of non-oncological diseases, the rationale for thecombination treatment in accordance with the present invention is alsothat there is a therapeutic advantage for the patient to combinespecific and mechanistically acting molecules with more broadly actingtherapeutic concepts. The expected effect of this combination is toavoid possible escape mechanisms for the target cells, to reduce therequired respective doses of the drugs in comparison to the doses usedin a mono-therapy (due to the additive or synergistic effect of thecombination), and to reduce the likelihood of the target cells todevelop resistances against the drugs.

LEGEND TO THE FIGURES

FIG. 1

Inhibition of VEGFR-2 phosphorylation after varying exposure of compoundMES(T) on NIH3T3 KDR cells. The upper panel shows a Western blot probedwith an antibody specific for phosphorylated tyrosine residues (α-PY).The lower panel shows a Western blot using an antibody specific forVEGFR-2 (α-KDR).

FIG. 2

Evolution of the tumour volume in nude mice bearing subcutaneous FaDutumours, untreated (dotted line), treated orally twice weekly with adose of 50 mg/kg of compound MES(T) (black line), or treated orallytwice weekly with a dose of 100 mg/kg of compound MES(T) (gray line).

FIG. 3

Evolution of the tumour volume in nude mice bearing subcutaneous ovariancancer SKOV-3 tumours, untreated (dashes), treated daily per os with 15mg/kg EGFR/HER2 inh. (triangles), treated daily with 50 mg/kg C12(T)(squares), or treated with the combination of 15 mg/kg EGFR/HER2 inh.and 50 mg/kg C12(T) (losanges).

1. A method of treating cancer diseases, which method comprisessimultaneous, separate or sequential administration of therapeuticallyeffective amounts of: (i)(Z)-3-(1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone,or a pharmaceutically acceptable salt thereof; and (ii)4-[(3-chloro-4-fluorophenyl)amino]-6-{[4-(N,N-dimethylamino)-1-oxo-2-buten-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)-quinazolineor a pharmaceutical acceptable salt the tautomers or the stereoisomersthereof; in the form of a combined preparation optionally adapted for aco-treatment with radiotherapy or radio-immunotherapy.
 2. The method inaccordance with claim 1 wherein (i) is the monoethanesulfonate salt of3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methoxycarbonyl-2-indolinone.3. The method in accordance with claim 1, wherein (ii) is thequinazoline derivative4-[(3-chloro-4-fluorophenyl)amino]-6-{[4-(N,N-dimethylamino)-1-oxo-2-buten-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)-quinazolineor a pharmaceutically acceptable salt thereof.
 4. The method inaccordance with claim 3, wherein (ii) is the di-maleic acid salt of thecompound4-[(3-chloro-4-fluorophenyl)amino]-6-{[4-(N,N-dimethylamino)-1-oxo-2-buten-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)-quinazoline.5. The method in accordance with claim 2, wherein (ii) is the di-maleicacid salt of the compound4-[(3-chloro-4-fluorophenyl)amino]-6-{[4-(N,N-dimethylamino)-1-oxo-2-buten-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)-quinazoline.6. The method in accordance with any of claims 1 to 5, wherein theformulation of (i) is for oral administration.
 7. The method inaccordance with any one of claims 1 to 5, wherein (i) is administered ina daily dosage such that the plasma level of the active substance liesbetween 10 and 500 ng/ml for at least 12 hours of a 24 hours dosinginterval.
 8. The method in accordance with claim 1 wherein the cancerdisease is selected from prostate cancer, renal cell cancers, bladdercancers, ovarian cancers, cervical cancers, endometrial cancers, lungcancer, colorectal cancers, pancreatic cancer, gastric cancer,oesophageal cancers, hepatocellular cancers, cholangiocellular cancers,head and neck cancer, malignant mesothelioma, breast cancer, malignantmelanoma or bone and soft tissue sarcomas, multiple myeloma, acutemyelogenous leukemia, chronic myelogenous leukemia, myelodysplasticsyndrome and acute lymphoblastic leukemia.
 9. The method in accordancewith claim 5 wherein the cancer disease is selected from prostatecancer, renal cell cancers, bladder cancers, ovarian cancers, cervicalcancers, endometrial cancers, lung cancer, colorectal cancers,pancreatic cancer, gastric cancer, oesophageal cancers, hepatocellularcancers, cholangiocellular cancers, head and neck cancer, malignantmesothelioma, breast cancer, malignant melanoma or bone and soft tissuesarcomas, multiple myeloma, acute myelogenous leukemia, chronicmyelogenous leukemia, myelodysplastic syndrome and acute lymphoblasticleukemia.
 10. The method in accordance with claim 9 wherein (i) isadministered in a daily dosage such that the plasma level of the activesubstance lies between 10 and 500 ng/ml for at least 12 hours of a 24hours dosing interval.
 11. The method in accordance with claim 8 or 9,wherein the cancer disease is selected from colorectal cancers.
 12. Themethod in accordance with claim 9, wherein the cancer disease isselected from colorectal cancers and wherein (i) is administered in adaily dosage such that the plasma level of the active substance liesbetween 10 and 500 ng/ml for at least 12 hours of a 24 hours dosinginterval.